Scientific Committee on Emerging and Newly Identified Health Risks
SCENIHR

Risk Assessment of Products of Nanotechnologies

The SCENIHR adopted this opinion at its 28th plenary on 19 January 2009

Risk Assessment of Products of Nanotechnologies

About the Scientific Committees
Three independent non-food Scientific Committees provide the Commission with thescientific advice it needs when preparing policy and proposals relating to consumer
safety, public health and the environment. The Committees also draw the Commission's
attention to the new or emerging problems which may pose an actual or potential threat.
They are: the Scientific Committee on Consumer Products (SCCP), the ScientificCommittee on Health and Environmental Risks (SCHER) and the Scientific Committee on
Emerging and Newly Identified Health Risks (SCENIHR) and are made up of external
experts.
In addition, the Commission relies upon the work of the European Food Safety Authority(EFSA), the European Medicines Evaluation Agency (EMEA), the European Centre for
Disease prevention and Control (ECDC) and the European Chemicals Agency (ECHA).
SCENIHR
Questions concerning emerging or newly-identified risks and on broad, complex or multidisciplinary issues requiring a comprehensive assessment of risks to consumer safety or
public health and related issues not covered by other Community risk-assessment
bodies.
In particular, the Committee addresses questions related to potential risks associatedwith interaction of risk factors, synergic effects, cumulative effects, antimicrobial
resistance, new technologies such as nanotechnologies, medical devices, tissue
engineering, blood products, fertility reduction, cancer of endocrine organs, physicalhazards such as noise and electromagnetic fields and methodologies for assessing new
risks.
Scientific Committee members
Anders Ahlbom, James Bridges, Wim De Jong, Philippe Hartemann, Thomas Jung, MatsOlof Mattsson, Jean-Marie Pagès, Konrad Rydzynski, Dorothea Stahl, Mogens Thomsen
Contact:
European Commission
Health & Consumers DG
Directorate C: Public Health and Risk Assessment
Unit C7 - Risk Assessment
Office: B232
B-1049 Brussels
Sanco-Sc1-Secretariat@ec.europa.eu© European Commission 2009(ISSN)The opinions of the Scientific Committees present the views of the independent scientists
who are members of the committees. They do not necessarily reflect the views of theEuropean Commission. The opinions are published by the European Commission in their
original language only.
http://ec.europa.eu/health/ph_risk/risk_en.htm

2

Risk Assessment of Products of Nanotechnologies
ACKNOWLEDGMENTS
Members of the working group are acknowledged for their valuable contribution to thisOpinion. The members of the working group are:
The SCENIHR members:
Prof. Jim Bridges
Dr. Wim De Jong (Chair and Rapporteur)
Dr. Thomas Jung
Prof. Konrad Rydzynski
External experts:
Dr. Jukka Ahtiainen1, Finnish Environment Institute, Finland
Prof. Ken Donaldson, University of Edinburgh, United Kingdom
Dr. Teresa Fernandes, Napier University, United Kingdom
Prof. Jorma Jokiniemi, Technical Research Centre and University of Kuopio, Finland
Dr. Wolfgang Kreyling, GSF-Research Centre for Environment and Health, Germany
Prof. Francelyne Marano, Université Paris Diderot – Paris 7, France
Prof. Dik van de Meent, University of Nijmegen, The Netherlands

The additionalacknowledged:

contribution

from

the

following

expertisgratefully

Dr Thomas Kuhlbusch, Institut für Energie- und Umwelttechnik IUTA e.V., Duisburg,
Germany

Declared Interest (see minutes of the 26th SCENIHR plenary meeting of 23 September 2008):
http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_mi_026.pdf

1

3

Risk Assessment of Products of Nanotechnologies
ABSTRACTThis Opinion deals with the recent developments in the risk assessment of nanomaterials
for both man and the environment. The in-depth characterisation of a manufactured
nanomaterial on the basis of its physical-chemical characteristics is essential. Due to thesize and material specific temporal evolution of some nanomaterials, potentiallyhazardous nanomaterials need to be characterised both ‘as manufactured’ and in thevarious possible forms ‘as delivered’ in biological systems, or to a human in a specificapplication, or to a particular ecosystem of concern. The characterisation ‘asmanufactured’ provides information for the material safety data sheet of the productitself. The characterisation ‘as used’ in biological systems is needed as properties ofnanomaterials may change considerably, notably the size distribution due toagglomeration/aggregation of the particles. An issue of specific importance is theproperties of the nanomaterial as it is actually used in products and to which consumers
may be exposed. For the risk assessment the latter characterisation is of highest
relevance.
Some specific hazards, discussed in the context of risk for human health, have been
identified. These include the possibility of some nanoparticles to induce protein
fibrillation, the possible pathological effects caused by specific types of carbon
nanotubes, the induction of genotoxicity, and size effects in terms of biodistribution.
Knowledge is gradually becoming available on the behaviour of manufactured
nanoparticles in the environment in terms of the development of possible fate scenarios.
For some nanomaterials, toxic effects on environmental organisms have been
demonstrated, as well as the potential to transfer across environmental species,
indicating a potential for bioaccumulation in species at the end of that part of the foodchain. Although for some manufactured nanomaterials adverse effects were observed.
they should not be extrapolated to other manufactured nanomaterials. Theseobservations indicate potential hazards which should be taken into consideration in thesafety evaluation of manufactured nanomaterials. As there is not yet a generally
applicable paradigm for nanomaterial hazard identification, a case by case approach forthe risk assessment of nanomaterials is warranted.
One of the main limitations in the risk assessment of nanomaterials is the general lack ofhigh quality exposure data both for humans and the environment. A differentiation
between background and incidental exposure is generally difficult in real life situations as
the methods employed mainly measure the presence of (nano)particles and do not
generally discriminate between the different types of particles (manufactured or naturally
occurring) that may be present. Currently, the risk assessment procedure for theevaluation of potential risks of nanomaterials is still under development. It can be
expected that this will remain so until there is sufficient scientific information available tocharacterise the possible harmful effects on humans and the environment. Therefore theknowledge on the methodology for both exposure estimations and hazard identificationneeds to be further developed, validated and standardised.

Keywords: nanomaterials, nanoparticles, hazard identification, risk assessment, humanand environment.
Opinion to be cited as:
SCENIHR (Scientific Committee on Emerging and Newly Identified Health Risks), Risk
assessment of products of nanotechnologies, 19 January 2009.

4

Risk Assessment of Products of Nanotechnologies
TABLE OF CONTENTSACKNOWLEDGMENTS ........................................................................................... 3
ABSTRACT .......................................................................................................... 4
EXECUTIVE SUMMARY........................................................................................... 7
1.BACKGROUND................................................................................................. 11
2.TERMS OF REFERENCE ..................................................................................... 12
3.Scientific Rationale .......................................................................................... 13
3.1. Introduction ............................................................................................. 13
3.2. Physical-chemical characterization and analysis............................................. 14
3.2.1.Characterisation of physical-chemical properties ................................... 15
3.2.2.Detection and analysis ...................................................................... 16
3.2.3.Nanomaterial preparations for biological testing .................................... 17
3.2.3.1.

The importance of dispersion ............................................. 17

3.2.3.2. Reference nanomaterials, characterisation and test item
preparation.................................................................................... 18
3.3. Developments in methodology to measure exposure...................................... 19
3.4. The interface between nanomaterials and biological systems........................... 21
3.5. Human health issues ................................................................................. 23
3.5.1.Nanoparticle-protein interaction.......................................................... 24
3.5.2.Toxicokinetics .................................................................................. 24
3.5.2.1.

General background.......................................................... 24

3.5.2.2.

Translocation of nanomaterials ........................................... 24

3.5.2.3.

Organ distribution after intravenous exposure ...................... 25

3.5.2.4.

Organ distribution after oral exposure ................................. 26

3.5.2.5.

Organ distribution after inhalation exposure ......................... 26

3.5.2.6.

Clearance of nanomaterials................................................ 28

3.5.2.7.

Conclusions on toxicokinetics ............................................. 29

3.5.3.Effects of carbon nanotubes ............................................................... 29
3.5.4.Genotoxicity .................................................................................... 31
3.5.5.Cardiovascular effects of nanoparticles ................................................ 33
3.6. Environmental issues ................................................................................ 33
3.6.1.Environmental fate and behaviour....................................................... 33
3.6.1.1.

General principles............................................................. 33

3.6.1.2.

Test methods for predicting environmental distribution .......... 36

3.6.1.3.

Test methods for degradation and transformation ................. 37

3.6.1.4.

Test methods for bioaccumulation....................................... 37

3.6.2.Bioavailability and exposure ............................................................... 38
3.6.2.1.

General Principles............................................................. 38

3.6.2.2.

Exposure to nanomaterials in experimental studies ............... 39

3.6.2.3.

Food chain effects and secondary poisoning ......................... 40
5

Risk Assessment of Products of Nanotechnologies
3.6.3.Environmental effects........................................................................ 40
3.6.3.1.

Environmental test systems ............................................... 40

3.6.3.2.

Methods of assessment in vitro .......................................... 42

3.6.3.3.

Methods of assessment in vivo ........................................... 42

3.7. NANOTECHNOLOGIES- RISK ASSESSMENT................................................... 45
3.7.1.Relevant physicochemical properties ................................................... 45
3.7.2.Read-across..................................................................................... 45
3.7.3.Development of the risk assessment framework.................................... 46
3.7.3.1.

Development of the SCENIHR algorithm .............................. 46

3.7.3.2.

Addressing deficiencies in the data base .............................. 46

3.7.4.Conclusion for the risk assessment...................................................... 48
3.8. Research needs ........................................................................................ 48
3.8.1.Characterisation of nanomaterials ....................................................... 49
3.8.2.Determination of human exposure ...................................................... 49
3.8.3.Identification of human hazards.......................................................... 49
3.8.4.Environmental exposure .................................................................... 50
3.8.5.Environmental hazards ...................................................................... 50
4.OPINION ........................................................................................................ 52
5.MINORITY OPINION ......................................................................................... 57
6.LIST OF ABBREVIATIONS ................................................................................. 58
7.REFERENCES .................................................................................................. 60

6

Risk Assessment of Products of Nanotechnologies
EXECUTIVE SUMMARY
Currently, the procedure for assessing the potential risks of manufactured nanomaterials
is still under development. It can be expected that this will remain so until there issufficient scientific information available to characterise the possible harmful effects onhumans and the environment. Therefore the knowledge on the methodology for both
exposure estimations and hazard identification needs to be further developed, validated
and standardised. As already detailed in previous SCENIHR opinions (SCENIHR 2006,SCENIHR 2007a), free and low solubility nanoparticles (nanomaterials) are a priority
concern in the context of human and environmental risk. It should be realised that(especially for inhalation exposures) exposure to particulate matter may be due tonatural and accidentally induced (i.e. combustion processes) nanoparticles.
For the characterisation of manufactured nanomaterials several issues are important. The
nanomaterial should be characterised as it is produced by a manufacturer, resulting in
information that may be used for safety evaluation and the material safety data sheet(MSDS) of the nanomaterial (nanoparticle) itself. In addition, the nanomaterial should becharacterised as it is used in biological systems for safety evaluation. Whennanomaterials come into contact with a biological fluid they may become coated withproteins and other biomolecules. The preparation of nanomaterials for use in biologicalsystems may considerably change nanomaterial properties, notably the size distribution
due to agglomeration/aggregation of the particles. Another issue is the characteristics ofthe nanomaterial as it is actually used in products, and to which consumers may be
exposed. For the risk assessment the latter characterisation is of highest relevance. A
consensus is now emerging regarding the physical-chemical properties that need to be
determined in the characterisation of nanomaterials and which properties may be
important in the risk assessment of nanomaterials. For (partially) soluble nanomaterials
the toxicity may be governed at least in part by the soluble species released from thenanomaterial. For low solubility or a slow release, the particulate nature of the substancemaybe relevant with regard to tissue distribution and local release of toxic species whichshould then be considered in the risk assessment of such nanomaterials.
There is a need for reference nanomaterials since this would allow the assessment of fate
and behaviour as well as effects, which could then be related to the material’s properties
and characteristics. It would also allow comparisons between different nanomaterials.
Some reference nanomaterials are available, but they are spherical model materials
which are certified primarily for size and are used mainly to calibrate instruments whichmeasure particle size. The absence of well-defined parameters to measure andstandardise test protocols is identified as a major obstacle for reference material
production.
Currently the definition of what is “nano” is still under debate. Generally nanomaterials
are defined as being smaller than about 100 nm in at least one dimension. The currently
proposed definitions use the size of the primary particle/structure as a starting point.
However, when a nanomaterial is in particulate form, the particles may be present assingle particles but might also be present as agglomerates/aggregates. Depending on thenanomaterial, the majority of the particulates may actually be agglomerates/aggregates.
This may lead to the misinterpretation that agglomerates/aggregates of nanoparticles
that have dimensions well beyond the 100 nm size are not considered nanomaterials. Yet
they retain specific physicochemical properties which are characteristic for nanomaterials,
most likely due to their relative large specific surface area (SSA). Therefore, whendescribing a nanomaterial it is important to describe not only the mean particle size but
also the size of the primary particles. In addition, information on the presence of
agglomerates/aggregates should be presented. When the mean particle size deviates(i.e. is larger) from the primary particle size this would indicate the presence of
agglomerates/aggregates. In addition to size the specific surface area as determined by
BET method is a good metric to describe particulates as this metric is independent of the
primary versus the agglomerated state. Hence, extending the current definition based on
physical size by the addition of a limit of the specific surface area to be above 60 m²/g of7

Risk Assessment of Products of Nanotechnologies
material volume (the value of 60 m²/g corresponds to the specific surface area of 100
nm solid spheres of unit density) should be considered.
One of the main limitations in the risk assessment of nanomaterials is the general lack ofhigh quality exposure and dosimetry data both for humans and the environment. One of
the issues is the difficulty in determining the presence of nanomaterials, and properly
measuring them on a routine basis in various substrates. In contrast to the situation forother exposure routes, for air-borne nanomaterials, analytical instruments are generally
available to determine exposure (size distribution of mass and number). This isparticularly true in the context of test atmospheres. However, differentiation between
background and incidental exposure is generally not possible in real life situations as themethods employed mainly measure the presence of (ultrafine) particles and do notdiscriminate between the different types of particles that may be present. There is a needto further establish reliable and standardised measurement techniques, to develop
measurement strategies, and to further implement screening/monitoring of nanoscaleparticles in sensitive work areas. Challenges are currently seen, especially in thedetection and assessment of manufactured nanoparticles in the environment. Similarly,
exposure estimates for consumers from food and consumer products remains difficult.
Information on the presence of manufactured nanomaterials solely relies on information(claims) provided by manufacturers. In addition, exposure estimation is also hampered
by lack of information on product use and use of multiple products containing
manufactured nanomaterials. In a similar fashion to air measurements, determination of
manufactured nanomaterials in consumer products suffers from the difficulty indiscriminating between background and intentionally added manufactured nanomaterials.
Coordinated efforts and research strategies for a comprehensive exposure assessment of
manufactured nanomaterials still have to be defined.
When nanomaterials contact a biological fluid they may become coated with proteins andother biomolecules. As the protein coating may affect the nanoparticle behaviour
including its biological effect, the nanoparticle may also have an effect on protein
behaviour. Nanoparticles were found to have the potential to promote and to retard
protein assembly into amyloid fibrils in vitro. These experiments were performed using
an incubation of nanoparticles with certain purified proteins. Whether the observed
nucleation process also occurs in an in vivo situation or in more complex biological fluids
where competitive binding may take place remains to be determined.
It should be noted that from the lung and gastrointestinal tract only minimal amounts(approximately 1% or less of the administered dose) enter the systemic circulation.
However, although minimal in percentage, this may result in a systemic availability of aconsiderable number of nanoparticles. The liver and the spleen are the two major organs
for distribution. For certain nanoparticles all organs may be at risk as, for all organs
investigated so far, either the chemical component of the nanoparticles or thenanoparticles themselves could be detected, indicating potential nanoparticle distribution
to these organs. These organs include the brain and the reproductive system (i.e. testis).
For distribution to the foetus in utero contradictory results were observed. The
knowledge on toxicokinetics has been increased showing that especially the smaller
nanoparticles do have a much wider organ distribution than the larger nanoparticles.
There are indications that after deposition at the olfactory mucosa of the nose
nanoparticles may translocate into the brain. This may offer a potential route of entry for
medicinal products into the brain. On the other hand this observation may also raisesome concern in view of the amyloid diseases of the brain in the context of the potentialof nanoparticles to cause protein fibrillation in vitro. This is certainly an area for which
additional research is urgently needed.
Based on the observations on the effects of particulate matter present in air pollution,some concern exists about the possible effect of manufactured nanoparticles on the
cardiovascular system. However, this has not been clearly demonstrated to be the casefor manufactured nanoparticles so far. Overall the information on the possible hazard ofnanoparticles for cardiovascular effects is rather limited and needs expansion.
8

Risk Assessment of Products of Nanotechnologies
When nanotubes were found to have similar characteristics to some types of hazardous
asbestos, it was demonstrated that similar inflammatory reactions can be induced bythese specific nanotubes as induced by asbestos. The main characteristics of substances
which induce these reactions are long thin fibrous forms (length >20 micrometer),rigidity, and no degradability (biopersistence). Whether such nanotubes would pose a riskfor humans is unknown, as in addition to these specific nanomaterial characteristics,
inhalation exposure to such structures would be essential. The main conclusion of thestudies on these specific carbon nanotubes relating to a risk for mesothelioma is thatsuch a risk cannot be excluded. So, when manufacturing nanotubes (possibly of any
chemical composition) one should be aware that certain characteristics (e.g. length,
rigidity, biopersistence) may pose a risk. The possibility for chronic inflammation andmesothelioma induction should therefore be considered in the safety evaluation of thatparticular manufactured nanomaterial.
The genotoxic effects of conventional particles are driven by two mechanisms – direct
genotoxicity and indirect (inflammation-mediated) genotoxicity. Nanoparticles may act
via either of these pathways since they cause inflammation and can also enter cells andcause oxidative stress. There is some evidence that the small size allows nanoparticles topenetrate into sub-cellular compartments like the mitochondria and the nucleus. The
presence of nanomaterials in mitochondria and the nucleus opens the possibility foroxidative stress mediated genotoxicity, and direct interaction with DNA, respectively. For
some manufactured nanomaterials genotoxic activity has been reported, mainly
associated to ROS generation, while for others contradictory results were obtained.
In view of the increasing production, use and disposal of manufactured nanomaterials,
there will be an increase in environmental exposure of these materials. As in the case ofhuman health risks, understanding the fate and behaviour of the manufactured
nanomaterials in the environment is crucial for predicting the potential ecotoxic effects invarious environmental species. Of major importance is the estimation of nanoparticle
release and fate, and exposures within the environment. For the environmental risk
assessment the estimation of water concentrations is essential. The assessment of
exposure concentrations of dispersed nanomaterials requires detailed insight into theprocesses that act on the particles in the environment. However, currently available
knowledge of these processes is insufficient to allow quantitative predictions of theenvironmental fate of nanomaterials.
The solubility of the nanomaterials is an important property that needs to be addressed.
Knowledge of the extent to which nanomaterials dissolve and the rate at which this takes
place is essential in two respects: (i) it is a direct control of the concentrations ofnanomaterials in the environment and of the time that the nanomaterials reside in theenvironment and in organisms, and (ii) it determines the concentrations of dissolved
species that originate from the nanomaterials. It is doubtful whether currently available
standard methods for measuring the (rate of) dissolution can adequately deliver thisknowledge.
Unlike in the assessment of exposure concentrations of conventional (dissolved) chemical
substances, the octanol-water partition coefficient Kow is likely to have a limited role inpredicting water-solids partitioning. An alternative theory to predict the exposure levelsof nanomaterials in water is yet to be developed. Based on well-established knowledge of
colloid science, it is expected that pH, ionic strength and presence of natural organic
matter in the water compartment (freshwater versus marine environments) are
important factors influencing the residual levels of nanomaterials in suspension.
Depending on these factors and the chemistry of the manufactured nanomaterial,
increased aggregation and thus sedimentation or in contrast enhanced dispersion may
occur.
In addition, for many manufactured nanomaterials the currently used methods (carbon
dioxide production, integration into biomass) for determining biological degradation will
not be applicable.

9

Risk Assessment of Products of Nanotechnologies
For some nanomaterials (i.e. quantum dots) the transfer across environmental species
was demonstrated indicating the potential for bioaccumulation via the food chain. For
simple organic chemicals, there is an established relationship between octanol waterpartition coefficient (Kow) and bioaccumulation or bioconcentration factor (BCF). However,
it is not known whether this relationship may be applicable for nanomaterials, asinsufficient data are available to evaluate this and more data are needed.
Ecotoxic effects on environmental species have been demonstrated, especially using
aquatic species. One of the major problems in ecotoxicological fate and effects testing is
the absence of consistent and broadly-applicable information on how nanomaterials areto be suspended or dispersed in various exposure media commonly used in
ecotoxicological testing. Mixing of nanomaterials with sediments/soils, as well as
characterisation over time, are areas which are still at a very early stage of development.
The common endpoints used in ecotoxicology such as mortality, growth, feeding, andreproduction can also be used for the evaluation of ecotoxicity by nanomaterials. In
addition, specific biomarkers similar to mammalian toxicity including oxidative stress,
genetic damage and gene expression may provide some insight in toxic mechanisms ofnanomaterials.
The health and environmental hazards were demonstrated for a variety of manufactured
nanomaterials. The identified hazards indicate potential toxic effects of nanomaterials for
man and environment. However, it should be noted that not all nanomaterials induce
toxic effects. Arguably, some manufactured nanomaterials have been in use for a longtime (carbon black, TiO2) and show low toxicity. The hypothesis that smaller means morereactive and thus more toxic cannot be substantiated by the published data. In thisrespect nanomaterials are similar to normal substances in that some may be toxic andsome may not. As there is not yet a generally applicable paradigm for nanomaterial
hazard identification, a case by case approach for the risk assessment of nanomaterials is
recommended.

10

Risk Assessment of Products of Nanotechnologies
1. BACKGROUND
Products of nanotechnologies are considered to bring benefits to everyday life of citizens
and to offer challenges for better optimisation of use of natural resources and protectionof the environment. They are already being marketed in sectors such as healthcare(targeted drug delivery, regenerative medicine, and diagnostics – as indicated by patent
analysis2), electronics, cosmetics, textiles, information technology, and environmental
protection. With the rapidly evolving process technologies, mass productions ofnanomaterials will take place implying also potential wide scale exposure of workers andconsumers as well as the environment.
The European Union has in its Strategy and Action Plan for nanosciences andnanotechnologies provided for developing the means to benefit from the potential of
nanotechnologies, but also to do this in a “safe, integrated, and responsible” way. A
review of the Community legislation in relation to nanomaterials was published. The
objective of safe, integrated and responsible development of nanotechnologies is also
pursued in the 7th Framework Programme for Research and Technological development
for 2007-2013, activities of the Joint Research Centres, national research programmes, inthe European Technology Platforms (ETPs) and research by industry and otherstakeholders. Internationally, the co-operation for the safety of nanotechnologies is also
taking place, especially with respect to activities in OECD, standardisation in ISO/CEN,
pharmaceutical products in Trans-Atlantic co-operation and for medical devices in the
Global Harmonisation Task Force.
In its opinion of 2006, the Scientific Committee on Emerging and Newly Identified Health
Risks (SCENIHR) concluded that nanomaterials may have different toxicological and(eco)toxicological properties than the substances in bulk3 form. Therefore their risks need
to be assessed on a case-by-case basis and the risk assessment methods andinstruments may require further development.
A second SCENIHR opinion, adopted on 21-22 June 2007, on the nanomaterials inTechnical Guidance Documents (TGDs) of chemicals legislation concluded that the current
methodologies described in the TGDs are generally likely to be able to identify thehazards, but modifications are required for the guidance on the assessment of risks tohuman health and the environment. Furthermore, the opinion highlights the need todetermine the appropriateness of current test procedures for the prediction of humanhealth hazards and estimation of risks for all types of nanoparticles/materials. Dependingon the regulatory environment, the roles and involvement of different parties andstakeholders, the scope and responsibilities for development and implementation of risk
assessment of nanomaterials vary across sectors/areas. It is therefore useful to
substantially contribute to a thorough exchange of scientific information acrosssectors/areas. Consequently, it is envisaged to either make use of existing, or organisingon a case-by-case basis, events or other suitable exchange mechanisms with allinterested parties to enhance exchange of the evolving scientific information from various
sources in the area of risk assessment of nanomaterials.
Hence, the Commission considers it important that this process benefits from, and is
supported by, the expertise that the Scientific Committees have built up in their opinions
over recent years. Therefore the SCENIHR is expected to update and provide scientificadvice on the risk assessment of nanomaterials in the light of new and upcoming2

See recent OECD-Document entitled “New patent analysis captures nanotechnology's current state ofdevelopment 15-Jun-2007”. This new STI Working Paper (2007/4) aims to capture current inventive activities
in nanotechnologies based on the analysis of patent applications to the European Patent Office (EPO) http://www.oecd.org/dataoecd/6/9/38780655.pdf.

3
In particle toxicology, the term “bulk” is often used to distinguish nanoparticles to larger particles of the samechemical substance. Equally relevant is the comparison of the nanoparfticulate form of a chemical with the free
(atomic, ionic, molecular) gaseous or dissolved species. All possible species (gaseous/dissolved, nanoform,
aggregates/agglomerates and conglomerates with other materials) may play a role in the way nanomaterials
affect organisms. In this text, the term “bulk” is used to refer to all non-nano species of a nanomaterial.

11

Risk Assessment of Products of Nanotechnologies
scientific information, including the outputs of various events on the Safety ofNanomaterials and the opinions of other Community Scientific Committees and groups,
including the European Group on Ethics (here especially: Opinion on Ethics ofNanomedicine), on substances by the European Chemicals Agency (ECHA), on food andfeed by the European Food Safety Authority (EFSA) and on pharmaceuticals by theEuropean Medicines Agency (EMEA). The scientific opinions will also provide inputs tovarious Commission activities. Based on these Commission activities a further
contribution to various activities at European and international level (i.e. in OECD,
ISO/CEN, and the EU-US Partnership activities) in the area of risk assessment of
nanomaterials is envisaged.

2. TERMS OF REFERENCEThe SCENIHR is asked:
To identify and assess new information and update the opinions of the SCENIHR on
potential risks of products of nanotechnologies, in particular, with respect tocharacterisation, eco-toxicology and toxicology as well as exposure assessments. Thisupdate should be done in a step-wise manner taking into account the upcoming risk
assessment demands related to specific nanomaterials and the evolving scientificinformation from various sources, including results from scientific research projects andactivities of the European Technology Platforms related to the safety of nanomaterials.
The update should:i)

Provide, on the basis of the results obtained, recommendations on:¾

improvements of existing test methods and/or on the development of newones, including in vitro and in vivo methods, to address aspects specific tonano in characterisation and hazard assessment;¾

improvements in exposure assessment (including, amongst others, also
relevant information on sampling, detection tests, instrumentation, modelling)
to address aspects specific to nano and provide a list of specificnanomaterials/particles with possible substantial exposure noting current
activities within the OECD Working Party on Manufactured Nanomaterials;¾

improvements in risk assessment in general including specifically information
linked to mechanistic information to address aspects specific to nano.

ii)

Recommend further prioritised needs for short, medium and long-term research inareas related to the possible risks of products of nanotechnologies based on aknowledge gap closure analysis.

iii)

Identify, as much as possible scientific evidence permits, direct or indirect healthrisks with regard to current and foreseeable applications of nanomaterials basedon information related to volume of production in different sectors. For the sector
of cosmetics and medical devices indications from patents4 should also specificallybe taken into account. Risks and specificities of different nanomaterials serving
the same purpose shall, in as much as possible, be compared.

It should be noted that the Commission may ask the SCENIHR and the SCCP to prepare
ad hoc opinions on specific applications of nanomaterials in the field of cosmetics andmedical devices and handle these as a matter of priority.

4
See recent OECD-Document entitled “New patent analysis captures nanotechnology's current state ofdevelopment 15-Jun-2007”. This new STI Working Paper (2007/4) aims to capture current inventive activities
in nanotechnologies based on the analysis of patent applications to the European Patent Office (EPO) http://www.oecd.org/dataoecd/6/9/38780655.pdf.

12

Risk Assessment of Products of Nanotechnologies
3. Scientific Rationale
3.1. Introduction
Over the last few years, there has been an increase in awareness of the potential risks
associated with manufactured nanomaterials. Legally, manufactured nanomaterials arecovered by the definition of substances5 as mentioned in the REACH legislation(Regulation (EC) No 1907/2006) (European Commission 2006). Risks associated with
substances have to be evaluated according to various EU regulations depending on
product category and production volume. A review of the European Community
legislation in relation to nanomaterials has recently been finalised (COM/2008/0366 final)(European Commission 2008). The main conclusion was that the current legislation does
cover in principle the potential health, safety and environmental risks in relation tonanomaterials. The protection of health, safety and the environment needs mostly to beenhanced by improving implementation of current legislation. In addition, it was
concluded that the knowledge on essential questions such as characterisation, hazards,
exposure, risk assessment and the risk management of nanomaterials needs to be
improved (European Commission 2008).
To date, the SCENIHR has published three Opinions dealing with various aspects of thepossible risks of the use of nanotechnology in all aspects of society. The first Opinion
dealt with the risk assessment methodologies available for evaluating the possible
adverse health and environmental effects of nanotechnology products (SCENIHR 2006),
while the second and third described more technical aspects on how to properly
investigate the safety of nanomaterials when using the Technical Guidance Documents
for the evaluation of dossiers of chemical substances (SCENIHR 2007a), and whatdefinitions within the nanotechnology area can be used for risk assessment (SCENIHR
2007b). It must be noted that nanotechnology has introduced new nanoparticulate formsof chemicals, of which properties, behavior and effects are largely unknown, and, hence,of concern. Although only two years have passed since the first evaluation of possible
risks of nanotechnologies, there has been substantial activity in the evaluation of theharmful effects of nanomaterials, notably in the evaluation of potential toxic effects ofnanomaterials by in vitro assays. Currently, in vitro assays are useful for screening
purposes and may provide valuable insights into the underlying mechanisms of adverse
effects. However, in vitro assays have their limitations, especially in relation to evaluationof a possible risk for humans and the environment. Therefore, at present, in vivo assays
are still needed for risk assessment.
An important issue of appropriate safety evaluation is the choice of an exposure dose in
the test system that is relevant for human or environmental nanomaterial exposure. In
addition, there are still some uncertainties about the best dose metric to be used insafety evaluations and the risk assessment of manufactured nanomaterials.
Another lack of current safety evaluation of nanomaterials is the fact that most in vitro
and in vivo studies are only short-term while impacts on human health and theenvironment are more likely to occur during and after long-term exposure. Consequently,
there is an urgent need for long-term exposure studies.
There are indications that there is a steady increase in products produced bynanotechnology or containing nanomaterials that are available on the market. The
inventory of the Woodrow Wilson International Centre for Scholars now contains almost
800 consumer products with a nanotechnology claim (WWICS 2008). A major drawbackof this registration is that it is based on voluntary information and claims from the
manufacturers, which in many cases cannot be verified.

5
Substance: a chemical element and its compounds in the natural state or obtained by any manufacturing
process, including any additive necessary to preserve its stability and any impurity deriving from the processused, but excluding any solvent which may be separated without affecting the stability of the substance orchanging its composition.

13

Risk Assessment of Products of Nanotechnologies
As a matter of example, one of the nanomaterials that has been increasingly applied isnanosilver, reported to be present in a great variety of products such as washingmachines, socks, food contact material, wound dressings and food supplements(Wijnhoven et al. 2009, WWICS 2009). The possible use of nanoformulations for foodsupplements must be looked at carefully as it may be regarded either as potentiallyhazardous (EFSA 2008) or as potentially beneficial depending on the specific case.
Increased bioavailability due to the nanoformulation of the supplement may be beneficial
for some applications but may create the possibility of overdosing.
In fact, when nanomaterials are firmly embedded in large structures, for example inelectronic circuits, they are less likely to escape this structure and no human orenvironmental exposure is likely to occur. However, while this may be true during
production and appropriate use of nanomaterial-containing products, exposure may occur
during abuse, waste and recycling. In other words without any exposure there is no risk.
Hence the estimation of exposure scenarios in terms of their frequency, their quantityand quality, and their targets (individuals, populations, etc.) are absolutely mandatory
for a rational risk assessment. It should be realised that (especially for inhalation
exposures) exposure to particulate matter may be due to natural and accidentallyinduced nanoparticles (i.e. particulate air pollution by combustion processes).
Based on discussions in OECD and ISO working groups, a consensus is now emerging on
the physical-chemical properties of nanoparticles that need to be addressed in the risk
assessment process of nanomaterials (OECD 2008a). It should be noted that theseproperties should also be determined for the nanomaterials as used in the testing forsafety evaluation, and not only on the nanomaterials as provided by the manufacturer.
For most nanomaterials, a full evaluation of potential hazards has not yet been
performed. Recently, the OECD has started a sponsorship programme in which, for 14 of
the most used nanomaterials, a dossier on hazard identification will be produced (OECD2008a). The programme contains an extensive list of endpoints to be determined
including those for nanomaterial information/identification, physical-chemical properties,
as well as material characterisation, environmental fate, environmental toxicology,
mammalian toxicology, and material safety (OECD 2008a). For this evaluation, current
OECD guidelines and other tests will be used. One of the outcomes of this programme
will be insights into the suitability of the current OECD guidelines for hazard identification
and where adaptations of these guidelines will be needed specifically for manufactured
nanomaterials. This will contribute to the design of a testing strategy.
This Opinion deals with the recent developments in the area of risk assessment of
nanomaterials. Some specific hazards have been identified which will be discussed in thecontext of risk for human health. These include developments in the understanding of
toxicokinetics of nanomaterials, the possibility of nanoparticles to induce protein
fibrillation, the possible pathological effects of specific types of carbon nanotubes,
genotoxicity and size effects. Knowledge is becoming available on the behaviour ofnanoparticles in the environment in terms of the development of possible fate scenarios.
In addition, effects on environmental organisms have been demonstrated. The staged
approach to the risk assessment of human and environmental risks as presented in aprevious Opinion of SCENIHR (SCENIHR 2007a) will be elaborated on further.

3.2. Physical-chemical characterization and analysis
There is a general need for harmonisation of the methodologies used for thecharacterisation of nanomaterials. As a starting point, the detailed description of thenanomaterials is very important to assess the physical-chemical properties of thenanomaterials with regard to their potential adverse effects. This should include a
description of the possible impurities or contaminants. Knowledge of the properties of thenanomaterials used is also necessary to be able to compare various studies. A consensus
14

Risk Assessment of Products of Nanotechnologies
is now emerging on which nanoparticle properties are important in the risk assessment of
nanomaterials (OECD 2008a). It should be noted that these properties should bedetermined for the nanomaterials both as used in the testing for safety evaluation and asprovided by the manufacturer.
Although nanomaterials themselves are covered by the definition of substance within the
REACH legislation (Regulation (EC) No 1907/2006) (European Commission 2006),
currently the definition of what is “nano” is still under debate. Various organisations haveproposed definitions of nanoscale using an upper limit of about 100 nm. It should be
noted that most currently proposed definitions use the size of the primaryparticle/structure as a starting point. However, when a nanomaterial is in particulate
form, the particles may be present either as single particles or as agglomerates oraggregates. Depending on the nanomaterial, the majority of the particles may even be
agglomerates or aggregates. This may lead to the misinterpretation that agglomerates oraggregates of nanoparticles that have external dimensions well beyond 100 nm are notconsidered nanomaterials. Yet they retain specific physicochemical properties
characteristic of nanomaterials, most likely due to their large specific surface area (SSA).
The uncertainty regarding the presence of nanomaterials (either determined by size,<100 nm, or SSA >60 m2/g when calculated for <100 nm unit density spheres) in
products becomes of major importance when the only information on the presence of ananomaterial relies solely on the information provided by the manufacturer. Currently, it
is frequently not possible to evaluate the nanomaterial contents of these products when
the nanomaterial in question is mixed into a complex matrix of the finished product. This
unresolved issue occurs in consumer products, particularly cosmetics and health careproducts, and also in food and feed products. All of these products contribute to thecurrent exposure of the European population.
When describing a nanomaterial it is therefore important to describe not only the mean
particle size but also the size of the primary particles. In addition, information on thepresence of agglomerates and/or aggregates should be presented. When the mean
particle size deviates (i.e. is larger) from the primary particle size this would indicate thepresence of agglomerates/aggregates. This information should be included in thedescription of the nanomaterial and/or the product containing the nanomaterial. In
addition to size, the specific surface area is a good metric to describe particulates. The
specific surface area as determined by the BET method (Brunauer et al. 1938) has theadvantage of being independent of the primary versus the agglomerated state.
Scientific toxicological data suggest that the total surface area of nanoparticles is areasonable metric to describe toxicological responses in biological systems. The total
surface area should not be confused with the specific surface area (SSA) where smaller
particles have a larger SSA independent of whether they are present as primary,
agglomerated or aggregated particles.

3.2.1.

Characterisation of physical-chemical properties

Discussions are currently ongoing, both at OECD and ISO level, concerning the various
characteristics of nanoparticles which need to be measured (OECD 2008a). The main
parameters of interest with respect to nanoparticle safety are:Physical properties• Size, shape, specific surface area, aspect ratio• Agglomeration/aggregation state• Size distribution• Surface morphology/topography• Structure, including crystallinity and defect structure• Solubility
15

Risk Assessment of Products of Nanotechnologies
Chemical properties• Structural formula/molecular structure• Composition of nanomaterial (including degree of purity, known impurities oradditives)• Phase identity• Surface chemistry (composition, charge, tension, reactive sites, physicalstructure, photocatalytic properties, zeta potential)• Hydrophilicity/lipophilicity
When nanomaterials are used in test systems, one has to be aware that some of theproperties which need to be determined are largely dependent on the surrounding media
and the temporal evolution of the nanomaterials. Thus, a primary focus should be toassess the nanomaterials in exactly the form/composition they have as manufactured,and in the formulation delivered to the end-user or the environment if the formulation
contains free nanoparticles. Nanomaterials can exist as nanopowders; suspended in air(ultrafine particles, nanoparticles, aerosols), suspended in liquid (colloids) andincorporated in solids. For biological safety evaluation, manufactured nanomaterials need
to be dispersed in an appropriate media. The interaction between these media and thenanomaterials can have a profound influence on the behaviour of the suspension.
With the increasing number of newly emerging manufactured nanomaterials theimportance of the potential dissolution kinetics needs to be emphasised. Since dissolution
kinetics is frequently proportional to the surface area, nanomaterials are likely to dissolve
much more rapidly than larger sized materials. This applies e.g. to silver nanoparticles
which are increasingly used for their release of silver ions as anti-bactericidal agents. Yet
the dissolution kinetics is not properly studied. The example of silver nanoparticles
highlights the complexity of risk estimates of nanomaterials since adverse interactions of
the silver nanoparticles with biological systems need to be distinguished from thoseinteractions of the ionic silver.
It should be emphasised that not all properties can be determined in every situation, nor
is it necessary to do so.

3.2.2.

Detection and analysis

Methods for the assessment of nanoparticles in the air (aerosols) and suspended inliquids or fluids have been further developed, and new methods have become available.
Notably, similar to most advanced chemical analysis many of these methods involve
research grade instruments requiring trained operating personnel and are not always
straightforward to use in typical ‘public health’ settings. On the other hand, mobile andportable/handheld equipment is also becoming available, and an increasing number ofstudies have been performed and published in recent years (Mordas et al. 2008, Smith
2004). However, the wealth of these studies relates to the background of atmosphericnanoparticles, and little work in the context of manufactured nanoparticles has actuallyappeared. Furthermore, there is still a deficiency of comparable, reproducible andrepeatable harmonised protocols for measuring and characterising nanomaterials(SCENIHR 2006). The ability to provide more routinely operable instruments, together
with optimised protocols is important for providing meaningful and valid data that arecomparable, reproducible, and repeatable, and which can produce a system of reliablerisk identification, assessment and management. This requires defining the metrics mostappropriate for hazard characterisation and exposure, including the methodology toperform the measurements. For a broader overview on the full portfolio of available
methods for nanoparticle detection and analysis, the reader is refered to SCENIHR(2006).

16

Risk Assessment of Products of Nanotechnologies
For the measurement of particles in air, a number of methods are available. They havebeen developped since the 1980’s, are very reliable and often highly sensitive, butsometimes costly. Depending on the physicochemical parameters of a nanomaterial andincluding off-line analyses, many companies provide instrumentation able to characterise
airborne particles down to the nanometer range. Experience is also available in the fieldof electron microscopy and microanalysis of nanoparticles in tissue sections andprecipitated on substrates (e.g. Geiser et al. 2005). Other measurement methods, inparticular optical techniques such as light scattering (Lindfors et al. 2004), can be applied
to suspensions in various gaseous and liquid media and to solid matrices. The particle
dynamics in suspension depend strongly on the medium of suspension.
Absorption and scattering microscopy of single metal nanoparticles allow for the trackingof nanoparticles suspended in the liquid phase (Van Dijk et al. 2006). This technology
resulted in new equipment with the capability of optical tracking and identification of
metal nanoparticles in fluids. The use of Condensation Nucleus Counters, well establishedin aerosol science, can now be routinely used to obtain information on nanoparticles, but
is still unable to discriminate the particles from the background.
In an analytical sense, the most powerful method, real-time single particle mass
spectrometry, has been further developed to provide a reliable method for theassessment of nanoparticles suspended in gases and liquids (by Electrospray Ionisation)with potential applicability to other fluids (Kane et al. 2001, Noble and Prather 2000).
Here, a mass spectrum suitable for chemical analysis of the components of individual
nanoparticles including the surface layer can be sampled and analysed. At least twocommercial set-ups are currently available. All these analytical techniques have their
specific reliability and sensitivity profiles and typically need to be combined to obtainreliable and specific assessments. Therefore, special consideration needs to be given toeach methodology to verify the characterisation of the nanomaterials in the various
phases. Typically for high performance analytical techniques, a number of generic issues
need to be considered in the application of these methods for a specific case (e.g.
accuracy, specimen preparation, role of substrate and presence of contamination).

3.2.3.

Nanomaterial preparations for biological testing

3.2.3.1. The importance of dispersion
When manufactured nanoparticles are analysed in a clean sample which does not containany other material, their physical-chemical properties can be studied (using the many
instruments which are commercially available) with the level of precision required fortheir targeted production and testing. However, if nanoparticles are mixed within amatrix of different materials, as is the case for scientific and technological applications,consumer products and in toxicological and ecotoxicological samples, then it becomes
exceedingly difficult to identify those nanoparticles since they may occur only in parts per
106 to 1012 of the surrounding matrix. In effect, the nanoparticles become “needles in the
hay stack” which are extremely laboursome to find, identify and characterise.
It is well known from colloid science that nanoparticles can form agglomerates oraggregates, especially when they are kept as powder under dry conditions. This tendency
to aggregate can create difficulties when testing the toxicity of nanoparticles. However,despite their tendency to aggregate, nanoparticles do not usually change their specificsurface area. The total surface area is an important parameter for interactions withbiological systems. Usually, a dry powder or a suspension in a water-based medium orsome other fluid is used to administer the nanoparticle into the biological system. Several
studies have made suggestions as to how best disperse the nanoparticles (Bihari et al.
2008, Buford et al. 2007, Sager et al. 2007). Best protocols may vary between the
different nanomaterials. It seems obvious that there should be a best attempt to render
the nanoparticle in a size that is relevant to the expected consumer/population exposure.

17

Risk Assessment of Products of Nanotechnologies
Dispersal methodologies suggested for particles using rational approaches include theuse of albumin, a fairly bland and ubiquitous globular protein (Bihari et al. 2008), andlung lining fluid phospholipids (Wallace et al. 2007). Researchers must be aware thatthese coatings may alter the properties of the nanomaterial being tested and, therefore,
the biological activity under consideration.
Synthetic detergents such as polyoxyethylene sorbitan monooleate (Wick et al. 2007)
and Tween (Warheit et al. 2003) have been used to disperse nanoparticles forexperimental purposes. Researchers must be aware that these additions may be toxic bythemselves or act as an antioxidant (e.g. Tween). These additions should be taken into
consideration when characterising the nanomaterials prepared for testing.
3.2.3.2. Reference nanomaterials, characterisation and test item
preparation"Reference material" (RM) is the generic name for the materials having a proven andsufficient homogeneity and stability in terms of a defined intended use. "Reference
substance" or "reference chemical" are terms used in toxicology for materials that needto meet similar conceptual requirements but that are used for hazard identification,
usually under GLP. Reference materials (RMs) need to be produced and used applying
the conditions and terms standardised and described in ISO guides 30 to 35. When used
in toxicology as test items, principles of OECD GD 34 and GLP apply mutatis mutandis(OECD 2005).
RMs can be used for different purposes, such as calibration, assessment of laboratory
proficiency or test method performance. In toxicological assays for hazard identificationreference substances/materials may be used for comparison with both positive (toxic)
and negative responses. Currently, a small number of reference materials already exist in
the field of nanomaterials and manufactured nanoparticles (e.g. gold nanoparticles fromthe National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA andsilica from the European Commission, Institute for Reference Materials andMeasurements (IRMM), Joint Research Centre (JRC), Geel, Belgium). They are sphericalmodel materials which are certified primarily for size and are used mainly to calibrate
instruments which measure particle size. The absence of well-defined parameters tomeasure ("measurands") and of standardised test protocols is identified as a major
obstacle for reference material production, because agreed and harmonised methods areneeded.
A typical issue of information generated by measurements or studies is to combine a
metrological part with knowledge about a reference material with an intended use intoxicological test systems under GLP. Toxicological test systems mimic routes (andscenarios) of exposure and typically require information about dosage. A study,
examination or test, when successfully performed, generates a prediction of the effect ofinterest. In toxicological tests, reliability AND relevance both contribute to the overall
predictive capacity and to the validity of a test for its purpose.
In practice, and in agreement with the requirements mentioned above, characterisation
results should be obtained and used in their appropriate context scenarios. The
information should be used for description of intrinsic and extrinsic properties. The
metrological principles of the reference nanomaterials available so far cannot be used or
extrapolated to toxicological tests and related results, but the information on theproperties provides a reliable basis as starting point for the test and reference items usedin such studies. The preparation and use of a reference material comprises two stages.
Stage 1 is the characterisation of the intrinsic properties of a reference nanomaterial, its
stability and homogeneity. Physicochemical properties need to be determined. The
physical state and preparation form of the material examined should thereby be relevant
for production and use. Sample preparation steps corresponding to analytical sample
preparation should be critically assessed with regard to being a determinant of themeasurement result itself.
18

Risk Assessment of Products of Nanotechnologies
When a reference material is prepared for use in test systems for toxicological evaluationor environmental fate analysis, it will be brought into a matrix/media/vehicle dependingon the type of test assays used. This comprises conditioning and choice of matrix
components. The prepared test sample should thereby correspond to the requirements ofthe test method and preferably be representative for the identified exposure situation.
Stage 2 comprises the characterisation of properties following test sample preparation.
Results depend on the protocol used and matrix components, which may be essential fora certain test system and form part of that test system. Several results may be gained
for the same reference nanomaterial and its properties depending on the conditioning
and matrix used.
Indeed, shape, size and surface area affect the hazards associated with nanomaterials, atleast because these parameters affect the transport properties of the particles(absorption, distribution, and excretion). Reference nanomaterials have to be seen in
their context of intended use. They are tools of Quality Assurance and method validation.
They serve method harmonisation and standardisation, and performance assessment.

3.3. Developments in methodology to measure exposure
One of the major routes of exposure for humans is considered to be inhalation, for which
a lot of information is available including exposure measurements. Exposure data are
available for non-manufactured nanoparticles (often referred to as ultrafines) fromcombustion processes but these data are not specific to manufactured nanoparticles.
Nevertheless, the knowledge gained from studies of combustion products may makeextrapolation possible and allow tentative conclusions to be drawn for nanoparticle
atmospheric transport and exposure in humans. In contrast to the situation for otherexposure routes, for airborne nanomaterials, analytical instruments are generally
available to determine exposure (size distribution of mass and number). This isparticularly true in the context of test atmospheres. However, it remains difficult todifferentiate background from incidental exposure in real life situations as these methods
mainly measure the presence of (ultrafine) particles and do not discriminate between the
different types of particles.
Exposure of humans and ecosystems may occur via the gas-, water-, and solid phases.
The latter may include food and consumer products such as cosmetics. The uptake route,dose, and group of humans exposed must be differentiated in addition to the exposure
matrix. For exposure measurements, three different groups are generally distinguished,
namely workers, consumers, and the general public. In the case of workers, inhalation is
generally the main route of exposure. In addition, consumers and the general public are
increasingly exposed to nanomaterials in various consumer products via oral and dermalroutesIt should be noted that part of the nanomaterials taken up by inhalation will result in agastro-intestinal uptake due to the mucociliary mechanism present in the lung forparticle removal. One key point, currently very often neglected in exposure and healtheffects studies, is the determination of the dose which can vary significantly. Taking
airborne exposure as an example, exposure to manufactured particles with a median
diameter of 90 nm leads to an overall internal dose of about 30-40% of the exposure
value while the same value for 20 nm particles increases to 70-80% (according to the
ICRP-model for a healthy worker) (ICRP 1994).
Figure 1 summarises the different measurement techniques and approaches possible forthe assessment of exposure. It also gives the outline to measurement strategies since it
differentiates personal and spatial (fixed point monitors) as well as continuous anddiscontinuous measurements. The limitations of measurement techniques directly
influence measurement strategies. Generally, quite a few measurement techniques are19

Risk Assessment of Products of Nanotechnologies
available to assess nanoparticles exposure including mass and number based techniques,
single particle chemical analysis online/offline techniques etc. (Kuhlbusch et al. 2008a).
The main handicap for making good exposure assessments is the lack of instrumentation
to determine personal exposure that can continuously analyse single particles or their
agglomerates/aggregates for chemical and physical properties relevant for health.

Figure 1: Exposure related measurements (adapted from Borm et al. 2006)
Only a few papers have been published on measurement strategies which are currently
necessary to allow first exposure assessments towards manufactured nanomaterials(Brouwer et al. 2004, Kuhlbusch et al. 2008b).
Currently most research and measurements have been conducted to assess the exposureof workers via inhalation. Data on airborne exposure are still scarce and do not always
clearly differentiate ambient from manufactured particles (Fujitani et al. 2008,;
Kuhlbusch et al. 2004, Kuhlbusch and Fissan, 2006, Kuhlbusch et al. 2008a, Kuhlbusch etal. 2008b, Maynard et al. 2004,Tsai et al. 2008, Wake et al. 2002, Yeganeh et al. 2008).
In most cases it was seen that agglomerates of nanoparticles with diameters >400 nm
are released during handling. In one case (Yeganeh et al. 2008), significant increases ofsub-100 nm particle number concentrations during the handling of carbonaceous
nanomaterials were reported. The latter indicates that coordinated measurement
campaigns in various work areas are still needed to derive a comprehensive overview.
No quantitative or qualitative measurements of manufactured nanomaterials in ambient
air outside of workplaces are known. Investigations by Murr (Murr et al. 2004, Murr and
Guerrero 2006) revealed that carbon nanotubes may originate from general combustion
processes and can be found in ambient locations. This illustrates the difficulty ofidentifying airborne manufactured nanomaterials.
Overall, the information base for exposure assessment in workplaces is currently built ona limited database which has to be improved in volume, comparability andreproducibility. This can be achieved by working on the feasibility of routine assessments,
developing reliable measurement techniques, standardising measurement techniques,developing measurement strategies and implementing the screening and monitoring of
nanoscale particles in sensitive work areas. Challenges are currently seen especially inthe detection and assessment of product nanoparticles in the environment.
In addition to particle size and number, other metrics can be determined to express
exposure. These include particle surface area, surface charge (zeta potential), surface
area reactivity (radical formation, photo-catalysis, oxidation/reduction) etc. The choice ofdose metrics depends on the endpoint of interest.
Exposure estimates from food and consumer products remains difficult. Information on
the presence of manufactured nanomaterials solely relies on information (claims)
provided by manufacturers. In addition, exposure estimation is also hampered by lack of
information on product use and use of multiple products containing manufactured
nanomaterials. In a similar fashion to air measurements, determination of manufactured

20

Risk Assessment of Products of Nanotechnologies
nanomaterials in consumer products suffers from the difficulty of discrimination between
background and intentionally added manufactured nanomaterials. Coordinated efforts
and research strategies for a comprehensive exposure assessment of manufactured
nanomaterials still have to be defined.

3.4. The interface between nanomaterials and biological systems
When nanomaterials come into contact with a biological fluid, the fluid usually penetrates
into pores of nanomaterials regardless of whether they are single particles or
agglomerate/aggregates. As a result, they may become coated with proteins (Blunk et al.
1993, Cedervall et al. 2007, Labarre et al. 2005) and other biomolecules. The coating
may then influence the outcome of the biological response to the nanoparticle. Proteins
have been the most widely studied in mammalian systems. The association anddissociation of proteins from the nanomaterials was found to depend on the particle
hydrophobicity and size (radius of curvature) (Cedervall et al. 2007). Many proteins
formed transient complexes with the nanomaterials, the binding and dissociation being
dependent on protein identity. Albumin and fibrinogen displayed relatively high rates ofboth association and dissociation compared to apolipoprotein A-I. When there is anexcess of biological fluid (serum) the lower abundance proteins with higher affinities may
even eventually dominate the proteins present on the particle surface, the so-called"protein corona" (Cedervall et al. 2007).

Original
nanomaterial

Changes due tointeractions in the
exposure medium

Agglomeration
due to storage

Changes due
to initialinteractions withbiological systems

Subsequent changes
affecting the interactions
with the target sites

Figure 2:

The potential changes in nature of a nanomaterial due to the surrounding
media

The body defences have evolved in such a way that they can deal with any conceivablekind of foreign material that enters the biological system, including bacteria, viruses andparticles. The foreign surface is coated with host molecules present at portals of entry.
These host molecules act in several ways. Some are opsonins and following binding toparticle surfaces, they are recognised by defence cells, which possess receptors for them,
resulting in phagocytosis and clearance e.g. scavenger receptors. The scavenger receptor
MARCO is required for lung defense against pneumococcal pneumonia and inhaledparticles (Arredouani et al. 2004). Proteins of the complement system bind to foreign

21

Risk Assessment of Products of Nanotechnologies
surfaces. This results in a cascade of effects including production of opsonins andinduction of inflammation. The complement system can also be involved in the response
to some dusts (Warheit et al. 1991). Immunoglobulins are present in serous fluids at
portals of entry. IgG is an opsosin that was found to modify the reponse to some
respirable fibres but not others (Donaldson et al. 1995). In addition, a number of hostproteins that are specific for certain portals of entry (e.g. Surfactant protein A) and nonspecific proteins bind to the particle surface (Kendall 2007). Fine airborne urban particles(PM2.5) sequester lung surfactant and amino acids from human lung lavage. The actualrole that these biomolecules play in the subsequent response is not known. However, it isprudent to consider that the outcome of the interaction of a particle with a biologicalsystem might depend on the coating that it receives and this has implications for in vitro
work and any situation where a particle is delivered into a biolgical system under nonphysiological conditions.
Inflammatory reaction is a key event that may occur following exposure to any solidmaterial, including nanomaterials. For several nanomaterials in vitro induction of
inflammatory cytokines was demonstrated (Carlson et al. 2008, Kim et al. 2003, Kocbach
et al. 2008, Zhang et al. 2008). Such inflammatory cytokines can also bind tonanomaterials (Kim et al. 2003). This may have implications when in vitro assays areused for the evaluation of the inflammatory properties of nanomaterials.
Important factors for the potential translocation/absorption of nanomaterial may be theprotein-nanomaterial interactions both in the lungs and in the gut. Upon contact withbody fluids, some nanomaterials have been found to interact with proteins andbiomolecules immediately (Linse et al. 2007, Lynch and Dawson 2008). This contact withbiological matrices (including food) may determine the behaviour of the nanomaterials, inaddition to the nature of the surface (charge, chemistry) itself, although this will ofcourse influence the binding of the biomolecules (Colvin 2003, Lundqvist et al. 2004, Nel
et al. 2006).
A recent systematic study of interaction of polystyrene nanoparticles with no modification(plain) or modification with positive (amine) or negative (carboxylic) charges indicatesthat the surface and the curvature (particle size) both influence the details of theadsorbed proteins, although in all cases, a significant fraction of the proteins bound werecommon across all particles (Lundqvist et al. 2008). Due to the curvature of thenanoparticle surface this may affect the tertiary structure of the binding protein resulting
in malfunctioning (Lynch et al. 2006). These nanoparticle protein interactions may not be
static but change during time (Cedervall et al. 2007). Such protein coatings on
nanoparticle may enhance membrane crossing and cellular penetration (John et al. 2001,
John et al. 2003, Panté and Kann 2002). This may even include crossing the nuclear
membrane as demonstrated for gold nanoparticle up to a size of 39 nm bound to the
nuclear core complex protein (Panté and Kann 2002).
A recent review has summarised much of the current state-of-the-art in proteinnanoparticle interactions (Lynch and Dawson 2008). However, there are several
complicating factors, such as the fact that the biomolecules surrounding thenanomaterial, sometimes referred to as "corona", are not fixed, but are in a dynamic
state. The corona equilibrates with the surroundings, with high abundance proteinsbinding initially, but being replaced gradually by lower abundance, higher affinity
proteins. This complicates the measurement of such a protein corona. A considerable
portion of the true biologically relevant biomolecules (proteins) will be associated with
the nanoparticles for a sufficiently long time that they are not affected by themeasurement processes – the so-called “hard-corona” (Lundqvist et al. 2008).
Additionally, changes in the biomolecule environment, such as uptake or biodistribution
will be reflected as changes in the corona. One may speculate whether the protein
determines the fate or distribution of the absorbed nanoparticles or the environment with
its specific proteins present. Indeed the coating of 500 nm polystyrene nanoparticles witha bioadhesive tomato lectin molecule did increase considerably the uptake of theparticles after oral administration (Hussain et al. 1997). The uptake in the GI tract of22

Risk Assessment of Products of Nanotechnologies
various polystyrene latex particles (ranging ion size from 50 nm to 3 microns) could be
increased or decreased by modification of the particle surface (Florence et al. 1995).
The significance of this for nano-safety and nano-risk assessment is clear, as it implies
that detailed characterisation of the nanoparticles in the relevant biological milieu is vital.
Evidence is emerging in the scientific literature that coating of nanoparticles with specificproteins can direct them to specific locations – apolipoprotein E for example has been
associated with transport of nanoparticles to the brain (Kreuter et al. 2002). Serum
albumin has been shown to induce uptake and anti-inflammatory responses inmacrophages, which were not present when the particles were pre-coated with surfactant
to prevent albumin binding (Dutta et al. 2007). In addition, coating with polyethylene
glycol (PEG) prevents the cellular uptake of nanomaterials and increases their half-life inblood (Niidome et al. 2006).

3.5. Human health issues
Many of the currently available OECD guidelines for the testing of chemicals are likely tobe adequate to detect potential hazards of manufactured nanomaterials as well
(SCENIHR 2007a). However, considering the particulate nature of the manufactured
nanomaterials some adaptation of the testing methodology is likely needed (SCENIHR
2007a). This is especially the case for the expression of the dose metric as administeredin the test systems. The currently available information in the use of the OECD guidelines
for the evaluation of manufactured nanomaterials is, however, limited. Warheit et al.
(2007) reported on a base set of toxicity tests for detection of the acute toxicity ofultrafine TiO2 particles using assays as described in various OECD guidelines. The resultsof most of the studies demonstrated low hazard potential in mammals or aquatic species
following acute exposures to the ultrafine TiO2 particle-types tested. In the studies
particle sizes were approximately 140 nm in diameter when TiO2 was dispersed in water,
but increased up to approximately 2000 nm when present in phosphate buffered saline(PBS), thus indicating the importance of nanoparticle characterisation as they are used invarious test conditions (see section 4.2). Recently, the OECD has started a sponsorshipprogramme in which, for 14 of the most used nanomaterials, a dossier on hazard
identification will be produced (OECD 2008a). In this programme the applicability of thevarious OECD guidelines for nanomaterial testing will also be evaluated.
In the possible applications of validated in vitro assays for determination of general toxic
effects of nanomaterials there has been almost no progress in relation to risk
assessment. There is still a clear need for validated in vitro assays for nanoparticle
evaluation. In the base set as reported by Warheit et al. (2007), only two in vitro assays
were used, both of which are assays for the detection of genotoxicity. One assay (Ames
test) uses bacterial cells and the other (chromosomal aberration test) uses mammaliancells. For the bacterial assays there can be reasonable doubt whether the manufactured
nanomaterials in the size range used (140 nm) can enter the bacterial cells. Recent
results reported by Sayes et al. (2007) did not support the use of in vitro assays for
toxicity endpoints. For five different particle types the range of toxicity end points
showed little correlation between in vitro and in vivo measurements for inhalation toxicity
profiles (Sayes et al. 2007). Recent evaluations of safety assessment of manufactured
nanomaterials indicated that in vitro assays may be useful, but mainly for screening andthe evaluation of specific mechanistic pathways (ECETOC 2006, Oberdörster et al.
2005a). So, they may be used for assessing the possible reactivity, inflammatory
potential and cellular uptake of nanoparticles. However, to be applicable in risk
assessment, these assays need to be validated and their relevance for in vivo hazard
identification needs to be demonstrated.
It should be noted that the in vivo assays as described in the various OECD guidelines
are not validated for nanomaterials either. However, the experience gained in the testing23

Risk Assessment of Products of Nanotechnologies
of chemicals with these assays indicates that they can be used for the detection of somepotential human and ecological hazards.
–
3.5.1.

Nanoparticle-protein interaction

As the protein corona may affect the nanoparticle behaviour including its biological effect,
the nanoparticle may also have an effect on protein behaviour. Nanoparticles were foundto have the potential to promote protein assembly into amyloid fibrils in vitro by assisting
the nucleation process (Linse et al. 2007). This phenomenon may have implications forhuman disease as protein self-assembly of a variety of proteins and peptides is known tocause human amyloid disease (Chien et al. 2004, Chiti and Dobson 2006, Koo et al.
1999). Large insoluble protein fibrils are formed resulting in amyloid plaques, an example
being dialysis related amyloidosis due to β2-microglobulin (Floege and Ehlerding 1996).
Various types of nanoparticles (copolymer nanoparticles, cerium oxide particles, quantum
dots, and carbon nanotubes) were found to enhance the probability of appearance of a
critical nucleus for nucleation of protein fibrils from human β2-microglobulin (Linse et al.
2007). The shorter nucleation phase depended on the amount and nature of the particle
surface. There was an exchange in protein on the particle surface in which β2microglobulin formed multiple layers on the particle surface providing a locally increased
protein concentration promoting oligomer formation. These results suggest a mechanism
involving surface assisted nucleation that may increase the risk for toxic cluster andamyloid formation (Linse et al. 2007). Further research demonstrated that besides anincrease in nucleation, nanoparticles may also retard the nucleation process which was
attributed to an effect on the nucleation step of the amyloid beta protein while theelongation step was unaffected (Cabaleiro-Lago et al. 2008). These experiments were
performed using an incubation of nanoparticles with purified β2-microglobulin protein.
Recently this effect of a shortening of the nucleation process was also demonstrated for
β-amyloid after incubation with TiO2 nanoparticles (Wu et al. 2008). Whether theobserved nucleation process also occurs in an in vivo situation or in more complex
biological fluids where competitive binding may take place remains to be determined.
There are indications that after deposition at the olfactory mucosa of the nose
nanoparticles may translocate into the brain (see below). This observation raises someconcern in view of the amyloid diseases of the brain in the context of the potential of
nanoparticles to cause protein fibrillation in vitro. This is certainly an area for which
additional research is urgently needed.

3.5.2.

Toxicokinetics

3.5.2.1. General backgroundToxicokinetics is the science dealing with absorption, distribution, metabolism andexcretion (ADME) of substances in the body. This whole cascade of events occurring afteran (external) exposure determines the internal exposure of organs at risk to potential
toxic substances. Prominent exposure routes of nanoparticles are inhalation, ingestionand skin uptake as well as intravenous injection for medical purposes.
3.5.2.2. Translocation of nanomaterials
Translocation of manufactured nanoparticles through the epithelium is likely to depend on
the physical-chemical properties of the nanoparticle, e.g. surface charge, hydrophobicity,
size, presence or absence of a ligand, and physiology of the organ of intake e.g. healthy
vs diseased state (where translocation may be increased or decreased depending on theillness) (Des Rieux et al. 2006). Under normal physiological conditions, paracellulartransport of nanoparticles would be extremely limited, as pore size at tight junctions is24

Risk Assessment of Products of Nanotechnologies
between 0.3-1.0 nm (Des Rieux et al 2006). Little is known on the behaviour and fate ofnanoparticles in the gastrointestinal tract (EFSA 2008).
While some mechanisms may be of general applicability for many biological membranes,
it must be noted that each membrane has specific tasks so that mechanisms related tothose tasks may not be applicable to other membranes. After translocation/absorption,
the distribution of nanoparticles inside the body over the various organ systems andwithin the organs needs to be determined. After the initial translocation/absorption of
nanoparticles the systemic circulation can distribute the particles to all organs and tissues
in the body.
As a model particle for nanotechnology research including toxicokinetic studies, metallic
colloidal gold nanoparticles are widely used. They can be synthesised in different forms(rods, dots), are commercially available in various size ranges, and can be detected at
low concentrations. Human cells can take up gold nanoparticles without cytotoxic effects(Connor et al. 2005). In particular for biomedical applications, they can be considered
relevant models, since they are used as potential carriers for drug delivery, imaging
molecules and even genes (Kawano et al. 2006), and for the development of novel
cancer therapy products (Hainfield et al. 2004, Hirsch et al. 2003, Loo et al. 2004, O’Neal
et al. 2004, Radt et al. 2004). In addition, gold nanoparticles have a history as labels for
tracking protein distribution in vivo in which proteins are coupled to small colloidal gold
beads at nanoscale dimensions (Heckel et al. 2004, Hillyer and Albrecht 1999).
3.5.2.3. Organ distribution after intravenous exposure
For systemic distribution, direct systemic exposure of organs can be obtained by the
intravenous route for which the total internal dose/exposure is equal to the administereddose. Distribution of particles occurs at multiple organs including liver, spleen, heart andbrain (De Jong et al. 2008, Ji et al. 2006).
When rats were intravenously injected with solutions containing various sizes of metallic
colloidal gold nanoparticles (10, 50, 100 and 250 nm), the distribution of gold
nanoparticles was found to be size-dependent, the smallest particles showing the mostwidespread organ distribution including blood, heart, lungs, liver, spleen, kidney, thymus,
brain, and testis (De Jong et al. 2008). The larger nanoparticles mainly resided in spleen
and liver. Intravenously injected gold nanorods (length 65 ± 5 nm; width 11 ± 1 nm)
accumulated within 30 min, predominantly in the liver. The PEGylation (coating with
polyethylene glycol) of these gold nanorods resulted in a prolonged circulation (Niidome
et al. 2006).
When negatively charged 1.4 nm or 18 nm gold nanoparticles were injected
intravenously, the 18 nm nanoparticles showed a similar pattern as described above withthe highest accumulation in liver and spleen. For the 1.4 nm gold nanoparticles only halfof the injected dose was present in liver and spleen, whereas the other half was found at
higher fractions in the other organs mentioned above, in soft tissues and in the skeleton.
Furthermore, the 1.4 nm gold nanoparticles were still circulating in the blood after 24hours (Semmler-Behnke et al. 2008). When the same gold nanoparticles were injected
intravenously in pregnant rats in their third trimester, both 1.4 nm and 18 nm particles
were found in the placenta and the foetuses (Semmler-Behnke et al. 2007). For 5 and 30
nm gold colloid solutions very small fractions were found to be transferred to the rat
fetus after intravenous administration (Takahashi and Matsuoka 1981). In contrast,
transfer to fetal tissue could not be demonstrated by Sadauskas et al. (2007) when gold
nanoparticles of 2 nm and 40 nm were injected in pregnant mice.
For intravenously administered TiO2 nanoparticles in rats with a dose of 5 mg/kg bodyweight and a size range 20-30 nm, the tissue content of TiO2 was determined 1, 14, and28 days after administration (Fabian et al. 2008). There were no detectable levels of TiO2
in blood cells, plasma, brain, or lymph nodes. The TiO2 levels (µg/g organ) were highest
in the liver, followed in decreasing order by the levels in the spleen, lung, and very low in
the kidney, and highest on day 1 in all organs. TiO2 levels were retained in the liver for25

Risk Assessment of Products of Nanotechnologies
28 days; there was a slight decrease in TiO2 levels from day 1 to days 14 and 28 in the
spleen, and a return to control levels by day 14 in the lung and kidney. A limitation ofthis study is that most of the particles administered were in the fine fraction up to 1 µm,
whereas only 10% by weight was in the nanosize range (<100nm).
The results obtained with studies using intravenous administration show that the main
target organs for particles are the spleen and liver which are abundant in phagocytic cells
i.e. macrophages and Kupffer cells. As was demonstrated for 10 nm particles, thesmallest nanoparticles may also show distribution to other organs besides the liver andspleen.
3.5.2.4. Organ distribution after oral exposure
Oral administration of metallic colloidal gold nanoparticles of decreasing size (58, 28, 10
and 4 nm) to mice resulted in an increased distribution to other organs indicating ahigher uptake with diminishing size (Hillyer and Albrecht 2001). The smallest particle (4nm) administered orally resulted in an increased presence of gold particles in kidney,
liver, spleen, lungs and even the brain. The biggest particle (58 nm) tested was detected
almost solely inside the gastrointestinal tract. For 13 nm sized gold colloids, the highest
amount of gold was observed in liver and spleen after intraperitoneal administration(Hillyer and Albrecht 1998). One might speculate whether such translocation of
nanoparticles is accompanied by transport of food components/molecules and thus may
create an (unwanted) port of entry, which may result in unexpected toxicity or otheradverse affects such as induction of allergy.
TiO2 particles of 500 nm were observed in all major tissues of the Gut AssociatedLymphoid Tissue (GALT) including Peyer’s Patches and mesenteric lymph nodes after
repeated oral administration by gavage for 10 days and evaluation at day 11 (Jani et al.
1994). Systemic exposure occurred as titanium and was detected by chemical analysis inblood, liver, lungs, spleen and heart. The presence of the TiO2 particles was confirmed byhistology in Peyer’s Patches, mesenteric lymph nodes, liver, spleen and lung. In the heart
the presence of particles could not be confirmed by histology. The highest levels Ti (µgper g tissue) were present in the lymphoid tissues like Peyer’s Patches, mesenteric lymph
nodes and the mesentery network. The colon with lymphoid tissue as the appendix anddiffuse lymphoid aggregates showed a high Ti level as well. It was concluded that the
500 nm TiO2 particle uptake was primarily taking place via the Peyer’s Patches (Jani etal. 1994).
In a 28 day oral toxicity study of silver nanoparticles (average 60 nm) a dose dependentaccumulation of silver was observed in all organs examined, i.e. blood, brain, kidneys,
liver, lungs, stomach and testes (Kim et al. 2008). The highest levels were observed in
the stomach, followed by kidney and liver, lungs, testes, brain and blood. In the mid andhigh dose treated groups all levels measured were significantly increased compared with
the non treated control group. The silver content was determined by atomic absorption
spectrophotometry (AAS) while histology for the confirmation of the presence of silver
particles was not performed. Silver levels in the kidneys were, for all doses investigated,
twice as high in female rats than in male rats which could not be explained by the resultspresented (Kim et al. 2008).
It can be concluded that for some nanoparticles the size may be a limiting factor in thepotential to cross the GI tract barrier, while for other nanoparticles a similar size may
result in uptake from the GI tract which may occur at sizes up to 500 nm.
3.5.2.5. Organ distribution after inhalation exposure
Several inhalation studies in rodents have shown the distribution of nanoparticles tonumerous organs including the liver, the spleen, the heart and the brain (Kreyling et al.
2002, Oberdörster et al. 2002,, Semmler et al. 2004; Semmler-Behnke et al. 2007).

26

Risk Assessment of Products of Nanotechnologies
The translocation of radiolabeled insoluble iridium nanoparticles was monitored after
inhalation (Kreyling et al. 2002). For both the 15 nm and for the 80 nm particles, most of
the particles remained in the lungs from which they were predominantly cleared via the
airways into the GI tract and the feces. After systemic uptake from the lungs minimal
particle translocation of <1% was observed to secondary organs such as the liver,spleen, heart and brain. The translocation of the 80 nm particles was about an order ofmagnitude lower than that of the 15 nm particles (Kreyling et al. 2002). Two studies(Semmler et al. 2004, Semmler-Behnke et al. 2007) report on long-term nanoparticle
biokinetics in secondary target organs over six months after a single short-term
nanoparticle inhalation. Only about 1% of the inhaled nanoparticles had crossed the airblood-barrier and accumulated in secondary target organs (liver, spleen, kidneys, heartand brain) as well as in the soft tissue and bone. After a transient maximum in all
secondary target organs between 1-2 weeks after inhalation, nanoparticle concentrations
remained surprisingly constant between week 3 and six months. Unexpected back-andforth trafficking was observed across the alveolar epithelium of the lungs of the 20 nm
sized iridium nanoparticles in the lungs of adult healthy rats after a single one-hour
inhalation (Semmler-Behnke et al. 2007). Although being retained in interstitial spaces
Ir-nanoparticle re-appeared on the epithelium during the next six months to be clearedby macrophage-mediated transport towards the mucociliary escalator and to GI-tract
after swallowing.
When negatively charged 1.4 nm or 18 nm gold nanoparticles were intratracheallyinstilled into rats, the distribution determined 24 hours later, showed a much higher
fraction (8% of instilled dose) of the 1.4 nm particles to be translocated into thecirculation and accumulated in secondary target organs than a 30-fold lower fraction of
18 nm particles (Semmler-Behnke et al. 2008). Particles were found in the liver, thespleen, the kidneys, the heart, the brain, the reproductive organs, soft tissue and theskeleton, but by far the highest fraction remained in the lungs. Furthermore, in one otherstudy for gold nanoparticles with sizes of 5-8 nm the majority of the inhaled
nanoparticles remained in the lung, although there was a very small but significant
fraction being translocated to the blood (Takenaka et al. 2006). When rats were exposedby inhalation for 5 days to gold nanoparticles (majority with a size <35 nm) gold was
only detected in the lung and olfactory bulb (Yu et al. 2007). After 15 days of exposure
gold could also be detected in other organs including heart, liver, pancreas, spleen,kidney and testis (Yu et al. 2007).
In mice exposed by inhalation to fluorescent Fe containing magnetic nanoparticles (size50 nm) for four weeks (4h/day, 5 days/week) the nanoparticles were distributed tovarious organs including liver, spleen, lung, testis and brain (Kwon et al. 2008). The
results indicated that both the blood brain barrier (BBB) and the blood testis barrier(BTB) were penetrated by the nanoparticles.
Rats were exposed to carbon nanoparticles (size 20-29 nm) labelled with stable isotope
C13 in a whole body inhalation chamber (Oberdorster et al. 2002). Only at high exposures
did the label start to accumulate in the liver 30 minutes after exposure. 18 and 24 h after
exposure the liver contained about five times more label than the lung. No significant
increase in label was observed in the other organs examined which included heart,
olfactory bulb, brain and kidney. It is worth noting that 13C is naturally present in eachorganism at a level of about 1%. Therefore, the deposited 13C labeled nanoparticles
represented less than the endogenous 13C content in the mouse lungs.
Ambient air particles and nanoparticles have also been demonstrated to translocate to
the brain after inhalation, and thus may potentially influence the central nervous system
Using 13C as model particles with a diameter of 36 nm translocation into the olfactory
bulb was indicated most likely originating from entry in the olfactory mucosa in the nose
(Oberdörster et al. 2004). Also for gold nanoparticles translocation to the brain wasobserved after inhalation exposure (Yu et al. 2007). When 15-20 nm iridium
nanoparticles were administered by inhalation up to 6 months after exposure iridium
could be detected in the brain (Kreyling et al. 2002, Semmler et al. 2004). Whether this27

Risk Assessment of Products of Nanotechnologies
was solely due to translocation via the nasal absorption is not certain as systemic
distribution was also observed in the study. For diesel exhaust containing a nanosized
fraction, inhalation exposure resulted in changes in brain activity as demonstrated bychanges in EEG signals (Crüts et al. 2008). These observations on possible directtranslocation of inhaled nanoparticles in the brain warrant further research to either
confirm or reject the hypothesis of nanoparticle association with various brain diseases.
In humans, most inhaled carbon nanoparticles remain in the lung (Brown et al. 2002,
Mills et al. 2006, Möller et al. 2008, Wiebert et al. 2006a, Wiebert et al. 2006b).
Translocation was found to be <1%. So, although the translocation of nanoparticles fromthe lungs may occur after inhalation, most nanoparticles remain in the lung and only aminute fraction may reach the circulation. Other efforts to study this translocation fromthe lung across the air-blood-barrier in humans failed because the experimental limits ofdetection were about 1% of the administered dose to the lungs and hence above thetranslocated fraction if at all existing (Brown et al. 2002, Mills et al. 2006, Möller et al.
2008, Wiebert et al. 2006a, Wiebert et al. 2006b). Studies using radiolabelled
nanoparticles may have their limitations as it is known that a dissociation of the label
from the nanoparticles may occur as shown for the radio-tracer 99mTc when used forlabelling carbon nanoparticles (Nemmar et al. 2002).
The results obtained with inhalation studies show that there is the potential of thesmaller nanoparticles to cross the air-blood barrier and enter the systemic circulation. In
general only a very small portion of the inhaled dose (< 1%) shows translocation. Itshould be noted that after inhalation exposure, systemic disctribution to other organs
may also be due to secondary gut uptake after removal of particulates from the lung by
the mucociliary mechanism. However, with repeated exposures and the low particle
fraction showing migration, it could mean that based on particle numbers a considerable
internal systemic exposure may occur.
3.5.2.6. Clearance of nanomaterials
A prominent clearance pathway of ingested nanoparticles is fecal excretion since every
nanoparticle which is not absorbed by the gut epithelium will leave the body via thispathway. Similarly inhaled nanoparticles which deposited on the airways of therespiratory tract will be transported by mucociliary action to the larynx from where it will
be swallowed entering the GI-tract. Furthermore, even insoluble nanoparticles depositedin the lung periphery (alveoli) may eventually be cleared by macrophage mediatedtransport to the distal end of the mucociliary escalator from where they are cleared as
described above. However, note that in the human body only 20-30% of the peripherally
deposited nanoparticles leave the lungs via this route (Kreyling and Scheuch 2000).
If nanoparticles enter the systemic circulation there are two potential clearance pathwaysfor excretion:
1
Glomerular filtration in the kidneys towards the bladder into urine. In fact, Choi etal. (2007) have observed that intravenously injected quantum dots with a size below 4.5
nm and not bound to any protein due to cystein surface modification will bequantitatively excreted in urine using a rat model. Furthermore, the same clearance
pathway into urine applied to intravenously injected, hydrophilically functionalised andpositively surface charged SWCNT and MWCNT of up to 2 µm in length was observed in aguinea pig model (Singh et al. 2006). Besides these studies 0.09 and 0.001% fractions of
intravenously injected gold nanoparticles of 1.4 and 18 nm size, respectively, wereexcreted in urine (Semmler-Behnke et al. 2008)
2.
Another potential pathway might be a hepato-biliary clearance of nanoparticles
from the liver via the bile into the intestine and feces. While this clearance pathway iswell known in pharmacology it is only postulated for nanoparticles.

28

Risk Assessment of Products of Nanotechnologies
3.5.2.7. Conclusions on toxicokinetics
Existing data show that nanoparticles can enter circulation from the respiratory tract or
the gastro-intestinal tract. These processes are likely to depend on the physical-chemical
properties of the nanoparticles such as size and on the physiological state of the organsof entry. The translocation fractions seem to be rather low; however, this is subject of
current intense research.
After the nanoparticles have reached the blood circulation, the liver and the spleen are
the two major organs for distribution. Circulation time increases drastically when thenanoparticles are hydrophilic and their surface is positively charged. For certain
nanoparticles all organs may be at risk as, for all organs investigated so far, either thechemical component of the nanoparticles or the nanoparticles themselves could be
detected, indicating nanoparticle distribution to these organs. These organs include thebrain and testis/the reproductive system. Distribution to the foetus in utero has also
been observed. As the knowledge of the long-term behaviour of nanoparticles is very
limited, a conservative estimate must assume that insoluble nanoparticles may
accumulate in secondary target organs during chronic exposure with consequences not
yet studied. There is a specific concern considering the possible migration of
nanoparticles into the brain and unborn fetus. Research in both of these areas has to be
conducted in order to either confirm or reject the hypothesis of nanoparticle association
with various brain diseases, and the possible reprotoxic effects of nanoparticles.

3.5.3.

Effects of carbon nanotubes

The superficial resemblance between carbon nanotubes and some other high aspect ratio(long thin) nanoparticles was commented upon early in concerns over the safety of
nanotubes (Donaldson et al. 2006, The Royal Society and The Royal Academy ofEngineering 2004). The term HARN, or High Aspect Ratio Nanoparticles, has been used tocover such structures. For fibrous-type or asbestos-like effects the major concern ismesothelioma, an unusual endpoint that is difficult to study but arises with unusual
specificity to certain fibre exposure. Because of the low rate of mesothelioma following
inhalation exposure in rats (a few % similar to the prevalence in exposed humans) direct
exposure of the peritoneal mesothelium by intraperitonal injection was developed as anassay in the eigthties (Miller et al. 1999, Pott 1995). Takagi et al. (2008) used thisapproach to examine the tumerogenicity of carbon nanotubes in p53-deficient mice.
Although Takagi et al. (2008) showed that both asbestos and carbon nanotubes caused
mesotheliomas whilst fullerenes did not the methodology was criticised (Donaldson et al.
2008, Ichihara et al. 2008) on several gounds including i) the presentation of the test
sample indicating that clumps of nanotubes were used that were hundreds of microns indiameter, ii) the dose of 3 mg that was injected into each mouse to obtain the dose of
109 fibres which was originally developed for rats but not mice and iii) the use of the p53
deficient mouse model for mesothelioma detection without using relevant controls.
Poland et al. (2008) used the mouse peritoneal model of direct mesothelial exposure.
They tested the acute inflammogenic effects of carbon nanotubes and their ability tocause granulomas on the surface of the diaphragm after one week. The acute response in
the peritoneal cavity, inflammation and granuloma formation, mimics the long-term
mesothelioma development (Davis et al. 1986, Kane 2006). Various controls were used
including long and short amosite asbestos samples that had been used in the 1980's andwhich produced mesotheliomas in the peritoneal cavity in the case of the long and nonein the case of the short fibers (Davis et al. 1986) Nanoparticullate carbon black,graphene in compact form, as opposed to graphene in a tubular form as in carbon
nanotubes, was also used. Two carbon nanotube test samples containing a fraction oflong straight nanotubes were compared with two samples comprised of short or tangled
nanotubes. Inflammation and granulomas were only found in the case of the longstraight nanotubes whilst the short/tangled nanotubes had no effect. This property of29

Risk Assessment of Products of Nanotechnologies
length-dependent inflammation and early fibrosis in a mesothelial exposure model wastherefore shared by both asbestos and the specific carbon nanotubes investigated.
This provides a first support for the contention that specific types of long carbon
nanotubes may be pathogenic, like hazardous asbestos, when they have similar
characteristics, such as length, rigidity and biopersistence. Whether this poses a risk forhumans would depend on whether there is inhalation exposure to these specific types ofcarbon nanotubes. In addition, the risk would also depend on the possibility for natural
migration of nanotubes to the pleural mesothelium from the airspaces. It needs sufficient
long straight CNTs to get airborne in workplaces to reach a threshold dose followed bytranslocation to the pleural mesothelium.
Pacurari et al. (2008) used mesothelial cells in culture and compared carbon nanotubes
to asbestos showing that carbon nanotubes induced activation of molecular signaling
pathways associated with oxidative stress, similarly to asbestos.
The above studies focus on the fibre paradigm for predicting carbon nanotubes effects.
Of course carbon nanotubes occur (probably predominantly) as tangled ‘particles‘ ofnanotubes material and not as ‘fibres’. So like a ball of string that can be very long yet fit
in the hand, carbon nanotubes can be long but be compactly tangled into particles. Thereis reason to think that such nanotubes pose a ‘particle-type’ hazard that is greater thanwould be anticipated and reviewed in Donaldson et al. (2006).
Extensive research on the effects of purified and non-purified single walled carbon
nanotubes on the respiratory tract of mice has been carried out at NIOSH, Pittsburg,
USA. The exposure of C57BL/6 mice to non-purified single walled carbon nanotubes (ironcontent of 17.7% by wt) at 5 mg/m3, 5 hr/day for 4 days was compared with pharyngeal
aspiration of varying doses (5-20 µg/mouse) of the same but purified single walledcarbon nanotubes. Both exposure regimens resulted in the development of multifocal
granulomatous pneumonia and interstitial fibrosis. Non-purified single walled carbon
nanotubes inhalation was more effective than aspiration of purified single walled carbon
nanotubes in causing inflammatory response, oxidative stress, collagen deposition andfibrosis as well as mutations of K-ras gene locus in the lung of C57BL/6 mice (Shvedova
et al. 2005, Shvedova et al. 2008a). Sequential exposure to the same single walledcarbon nanotubes and bacteria enhanced pulmonary inflammation and infectivity(Shvedova et al. 2008b). In in vitro studies on a RAW 264.7 macrophage cell line thesame purified and non-purified single walled carbon nanotubes showed that the presenceof iron in single walled carbon nanotubes may be important in determining redoxdependent responses of macrophages (Kagan et al. 2006)
When nanotubes, possibly of any chemical composition, have similar characteristics assome types of hazardous asbestos, it was demonstrated that similar inflammatory
reactions can be induced by the nanotubes as asbestos. The main characteristics
required for this to occur are long thin fibrous forms (length >20 micrometer), rigidity,
and non-degradability (biopersistence). Whether such nanotubes would pose a risk forhumans is unknown, as besides these specific nanomaterial characteristics, inhalation
exposure to such structures would also be essential. In addition, migration of such
fibrous nanomaterials from the airspaces in the lung to the pleural mesothelium has tooccur. In terms of occupational safety, the local air concentration also needs to be higher
than threshold doses. The main conclusion of the studies on carbon nanotubes relating toa risk for mesothelioma is that such a risk cannot be excluded. So, when manufacturing
nanotubes (possibly of any chemical composition) one should be aware that certain
characteristics may pose such a risk and thus should be considered in the safety
evaluation of that particular manufactured nanomaterial. Carbon nanotubes seem toconform to the same paradigm as some forms of asbestos, glass fibres etc., that any
long, thin biopersistent fibre poses a potential mesothelioma hazard. This means that
other high aspect ratio nanoparticles such as nanowires or nanorods are likely to havethe same hazard if they satisfy the criteria of length and biopersistence.

30

Risk Assessment of Products of Nanotechnologies
3.5.4.

Genotoxicity

The genotoxic effects of conventional particles are driven by two mechanisms – direct
genotoxicity and indirect (inflammatory processes-mediated) genotoxicity, as reviewed
by Schins et al. (2007). Nanoparticles may act via either of these pathways since they
may cause inflammation (see above) and they can also enter cells and cause oxidative
stress (Donaldson et al, 2005, Nel et al. 2006, Oberdörster et al. 2005a, Oberdörster et
al. 2005b, Stone et al. 2007). There is some evidence that the small size may allow
nanoparticles to penetrate into sub-cellular compartments that normally exclude
environmental particles, like the mitochondria, and nucleus (Chen and von Mikecz 2005,
Li et al. 2003, Geiser et al. 2005). The presence of nanomaterials in both mitochondria
and the nucleus opens the possibility for oxidative stress mediated indirect genotoxicity,and direct interaction of nanoparticles with DNA and histones. Besides oxidative stress,
additional mechanisms of genotoxicity which may be specific for nanomaterials also need
to be considered, such as possible mechanical interferences during cell division, andother sources of genotoxic effects (i.e. metal release by nanomaterials) (Gonzalez et al.
2008).
Several studies with nanoparticles have indicated that some nanoparticles may be
genotoxic (reviewed by Gonzalez et al. 2008, Landsiedel et al. 2008). The mostfrequently used test was the Comet assay demonstrating the presence of DNA damage.
For several nanomaterials a positive outcome on genotoxicity was observed including C60
fullerene, single walled carbon nanotubes (SWCNT), nanoparticles of cobalt chrome(CoCr) alloy, TiO2, nanosized metal oxide V2O3, Carbon Black (CB), and nanosized diesel
exhaust particles.
The second most frequently used assay was the micronucleus assay in which thepresence of micronuclei in dividing cells is indicative for chromosomal aberrations. In the
micronucleus assay positive results were obtained for nanoformulations of TiO2, SiO2,
CoCr, ZnO and multi-walled carbon nanotubes (MWCNT). For the gene mutation assays
some studies showed a positive result for several nanomaterials including nano-FePt,
SiO2, TiO2, MWCNT, and CB.
For all three assay systems used (Comet, micronucleus and gene mutation), negative
results were obtained for TiO2, CB, SiO2, and single walled carbon nanotubes, while forsome nanomaterials contrasting results were obtained (Landsiedel et al. 2008). Theinterpretation of the data presented in the reviewed papers was hampered by various
limitations including the differences in the methodology used within one assay type, theuse of non-standardised methods with different primary cells or cell lines, and by thesometimes minimal characterisation of the nanoparticles tested and the lack of
information on possible contaminants.
For TiO2 and CB it was reported that the smaller (~20 nm) particles induced DNA
damage while larger particles (~200 nm) did not (Gurr et al. 2005, Mroz et al. 2008,
Rahman et al. 2002). Cobalt nanoparticles have been shown to induce more DNA
damage than micron sized particles using human fibroblasts in tissue culture in thealkaline comet assay (Papageorgiou et al. 2007). In the micronucleus assay Co
nanoparticles showed minor changes, whereas in the Comet assay for the same Co
nanoparticles, clear statistically significant positive results were observed (Colognato etal. 2008).
Some studies showed that highly purified amorphous silica, with a low surface reactivity,
was negative in the Comet assay (Barnes et al. 2008). This might suggest that
nanoparticles with low surface reactivity are likely to be less genotoxic than others. In
addition to the negative results of Barnes et al. (2008), very mild positive results (Yanget al. 2009) on DNA damage in Comet assay were reported after exposure of mouseembryo fibrolasts 3T3 to different concentrations of SiO2 nanoparticles (size 20 – 400nm). In two types of genotoxicity assays i.e. the micronucleus assay and the genemutation assay positive results were observed for silica nanoparticles (Landsiedel et al.
2008) while in the Comet assay weak positive results were obsreved (Yang et al. 2009).

31

Risk Assessment of Products of Nanotechnologies
A variety of genotoxicity (Ames test, clastogenicity in mammalian cells) and photogenotoxicity (Photo-Ames test, photo-clastogenicity in mammalian cells) tests have been
performed under GLP conditions on 14 different sunscreen-grade TiO2 (anatase andrutile; coated an uncoated; particle size range 11-60nm + one pigment grade – 200 000nm). All results were negative. They were provided as an unpublished industry safety
dossier but reviewed, summarised and published in the Opinion of the ScientificCommittee on Cosmetic Products and Non-Food Products Intended for Consumers
Concerning Titanium Dioxide (SCCNFP, 2000). Negative photo-clastogenic results werealso found in chromosome aberration tests on Chinese hamster ovary cells with a variety
of TiO2 particles (anatase, rutile; particles size: 14-60 nm) (Theogaraj et al. 2007).
However, others (Rahman et al. 2002, Wang et al. 2007) documented that ultrafine TiO2
particles increased the number of micronuclei in Syrian hamster embryo cells and ahuman B-cell lymphoblastoma (WIL2-NS) cells. In the latter model mutation frequency
was increased in the HPRT test and DNA damage was indicated by the Comet assay.
Positive results in micronucleus test and oxidative DNA damage were found recently infish cell lines derived from rainbow trout and goldfish skin (Reeves et al 2008, Vevers
and Jha 2008). It was suggested that several types of TiO2 (anatase; particle size of 255– 420 nm) were not genotoxic but photo-genotoxic in mouse lymphoma and Chinese
hamster lung cells (Nakagawa et al. 1997). This was further supported by the study of
Dunford et al. (1997) which showed DNA oxidative damage in human fibroblasts (MRC-5)
using the Comet assay. Similar to silica (SiO2) positive results were observed for TiO2 in
all three types of genotoxicity assays (Landsiedel et al. 2008).
Inconsistent results were published for the genotoxicity of zinc oxide nanoparticles(Brayner 2008, Dufour et al. 2006, Nohynek et al. 2007, SCCNFP 2003, Yang et al.
2009).
It was shown that silver nanoparticles (ca. 60 nm size) did not increase micronuclei
formation in rat bone marrow in an in vivo 28-days oral exposure test in doses up to
1000 mg/kg (Kim et al. 2008).
The genotoxicity of C60 has been relatively well studied, but conclusions are conflicting.
Dhawan et al. (2006) demonstrated a weak genotoxicity of colloidal C60 in Comet assay
performed on human lymphocytes. Several types of carbon nanomaterials such as carbon
nanoparticles that generate reactive oxygen species (ROS) were found to be genotoxic(Jacobsen et al. 2008). On the other hand, Zakharenko et al. (1997) reported no
genotoxicity of C60 in concentrations as high as 450 µg/l in SOS Chromotest and slightgenotoxicity (at 2.2 mg/l) when somatic mutation and recombination test in Drosophilamelanogaster was used. Mori et al. (2006) obtained negative results for fullerenes inboth a bacterial Ames test and mammalian cell chromosomal abberation tests.
In order to consider a specific engineered nanomaterial genotoxic, confirmation by two
independent laboratories and in two test systems is necessary and minimal criteria
should be met as proposed by Gonzalez et al. (2008). There are various inconsistencies
in the results reported so far (reviewed by Gonzalez et al. 2008, Landsiedel et al. 2008).
A major limitation in concluding whether a certain nanomaterial is genotoxic or not is the
scarce description and minimal characterisation of the nanomaterial samples used in thevarious studies.
In conclusion, for some manufactured nanomaterials, in vitro genotoxic activity has beenreported, but negative contradictory results were also obtained, and not all results could
be confirmed by in vivo testing. One potential cause of inconsistencies is the difficulty indelivering the nanomaterials to the test systems appropriately. Most available in vitro/in
vivo genotoxicity studies have been performed at high particle concentrations. In in vivo
situations, this may be associated with marked inflammatory and proliferative responses,
and hence may obscure and/or modify genotoxicity and even carcinogenicity readouts. In
addition, various assays with different primary cells and cell lines were used which did
not always show consistent results. Such inconsistencies may depend on physicochemical characteristics of the test material investigated such as size, shape,
32

Risk Assessment of Products of Nanotechnologies
aggregation/agglomeration state, surface properties, contaminants present and the cell
type used.
3.5.5.

Cardiovascular effects of nanoparticles

Air pollution is increasingly recognised as an important factor for cardiovascular diseasein urban communities (reviewed by Mills et al. 2009). The occurring cardiovascular
events including myocardial infarction and heart failure were attributed to the exposure
to combustion derived nanoparticles that incorporate reactive organic and transition
metal components. It was suggested that after inhalation the resulting respiratory
inflammation induces systemic effects either directly by translocation from the lung or
indirectly by yet unknown mediators (Mills et al. 2009). In view of these findings also for
manufactured nanomaterials a risk for interaction with the cardiovascular system can be
imagined.
Varous types of carbon nanomaterials were investigated and compared to a standard
urban particulate matter by Radomski et al. (2005). Mixed carbon nanoparticles, single
wall carbon nanotubes, multi wall carbon nanotubes, but not C60 fullerenes were found to
stimulate platelet aggregation in vitro, and to accelerate vascular thrombosis in a ferricchloride model of thrombosis in a specific rat model. No information was presented on
the characteristics of the carbon nanomaterials used, which limits the value of theobservations. In a recent study, modified fullerenes (C60(OH)24) were found to facilitate
adenosine diphosphate (ADP)-induced platelet aggregaton in vitro, whereas C60(OH)24
alone or carbon black did not (Niwa and Iwai 2007).
In contrast for several nanomaterials designed for drug delivery purposes, no or limited
effects on platelet function in vitro was noted including alcohol/polysorbate nanoparticles(Koziara et al. 2005), gadolinium nanoparticles (Oyewumi et al. 2004), andnanostructured silica hydroxyethyl methacrylate biocomposites (Liu et al. 2008).
Based on the observations some concern exists on the possible effect of manufactured
nanoparticles on the cardiovascular system. However, so far this has not been clearly
demonstrated to be the case for manufactured nanoparticles as well. Overall theinformation on the possible hazard of nanoparticles for cardiovascular effects is rather
limited and needs expansion.

3.6. Environmental issues
Inevitably, production, use and disposal will lead to releases to the environment.
Wastewater treatment streams, landfill and combustion of products containing
nanomaterials are means through which they may end up in the environment, although it
is most likely that they do so as modified forms from their primary counterpart. In
addition, some nanomaterials are used in environmental remediation applications and assuch they are applied as primary nanomaterials to the environment.

3.6.1.

Environmental fate and behaviour

3.6.1.1. General principles
The environmental fate and behaviour of nanomaterials has been recently reviewed byKlaine et al. 2008. Knowledge from colloid science can provide information on the likely
fate and behaviour of nanomaterials (Lead and Wilkinson 2006). The behaviour ofnanoparticles in the environment is expected to depend not only on the physical andchemical character of the nanomaterial, but also and perhaps predominantly on thecharacteristics of the receiving environment (Chen et al. 2008, Chen and Elimelech 2008,
33

Risk Assessment of Products of Nanotechnologies
Saleh et al. 2008). It is generally known that small particles tend to aggregate or
agglomerate to eventually become associated with other dissolved, colloidal andparticulate matter present in the environment. Upon entry into the environment,
nanoparticles may remain intact or undergo one or more of the following:•

dissolution,•

speciation (i.e. association with other ionic or molecular dissolved chemical
substances),•

biological or chemical transformation to other chemicals, and/or complete
mineralization (to carbon dioxide and water),•

agglomeration/disagglomeration,•

settling.

So far, there are no peer reviewed publications providing information on concentrations
or amounts of nanomaterials in environmental compartments such as surface waters andsoils. Estimates of quantities of nanomaterials present in surface waters and other media
derive from calculated exposure scenarios based on predicted nanomaterial use and notfrom actual measurements (Boxall et al. 2007, Mueller and Nowack 2008). Methods formeasuring nanomaterials in specific environmental matrices are being developed forvarious materials (Christian et al. 2008, Hassellöv et al. 2008, Tiede et al. 2008). The
appropriate metrics of the measurement of manufactured nanomaterials in relation toenvironmental risk assessment is still under discussion.
In order to assess the potential effects of nanomaterials in the environment, exposure
concentrations or doses should be considered realistically. All forms, in which thenanomaterial occurs, not only the free nanoparticulate form, but all physical andchemical species, should be considered. It is important to realise that there may be some‘hot spots’ where nanomaterials might concentrate due to their tendency toaggregate/agglomerate and potentially to adsorb to or associate with organic matter. In
addition, it is likely that some of the nanomaterials going through the standard waste
treatment stream will end up associated with the solid phase and then potentially bedeposited in certain areas of the environment where they might reach higher loads.
In the environment nanomaterials are expected to occur mainly associated withsediments and soils (Baalousha et al. 2008, Klaine et al. 2008). The free dispersed formof nanomaterials is of particular importance and is addressed here specifically. It is
recognised that for the estimation of the possible presence of free nanoparticles
knowledge on release scenarios is very important. Unfortunately, to date, there is nosuitable information available on this topic. Examples of exposure routes fornanomaterials via the environment are inhalation by humans, and other air-breathing
species, and uptake by aquatic organisms from water or sediments.
Assessment of exposure concentrations of dispersed nanomaterials requires detailed
insight into the processess that act on these materials in the environment. However,currently available knowledge of these processes is insufficient to allow quantitative
predictions of the environmental fate of nanomaterials.
Air
Information concerning the presence of nanomaterials in air is summarised in section
4.3.
Water
Upon release to water, dispersed nanomaterials are expected to behave according towell-understood phenomena described and explained in colloid science (Jones 2002,
Lyklema 2005). Colloidal suspensions of nanomaterials are generally expected to beunstable: i.e. upon collision, particles may approach each other close enough for

34

Risk Assessment of Products of Nanotechnologies
attractive Van der Waals forces to become dominant over repulsive electrostatic forces
and steric hindrance. As a consequence, particles adhere to each other and then settle
due to gravity (Baalousha et al. 2008, Ju-Nam and Lead 2008, Saleh et al. 2008).
Moreover, natural waters contain many other dissolved, colloidal and solid materials(including natural nanomaterials) to which nanomaterials can and usually will adhere.
Suspensions of dispersed nanomaterials are stable only under narrow ranges ofenvironmental conditions (Baalousha et al. 2008).
The dominant factors in colloid stability under natural conditions are known to be pH,
ionic strength and presence of natural organic matter (Lead and Wilkinson 2006). In sea
water with high pH and ionic strength, electric double layers of colloid particles are muchsmaller than in freshwater, allowing for closer interparticle approach, which usually leads
to more aggregation. In addition, the intrinsic properties and characteristics of thematerials, including their specific chemistry, will influence their fate and behaviour. The
surface properties of the nanomaterials are very important for their aggregation
behaviour, and thus for their mobility in aquatic and terrestrial systems, and as such fortheir interaction with and general bioavailability to organisms.
The humic and fulvid acids of "brown waters" will cover the nanoparticles with a coatingthat keeps them probably longer and more dispersed (Hyung et al. 2007). For example,
the presence of natural organic matter (NOM), as well as iron oxide, have a stabilising
effect on aquatic suspensions of fullerene and carbon nanotubes, at least in fresh water
systems (Baalousha et al. 2008, Chen and Elimech 2008, Christian et al. 2008, Saleh etal. 2008). A similar effect has been shown recently for nanoparticles of CeO2 (Quik et al.
2008). On the other hand, Baalousha et al. (2008) have shown that the effects of humic
acids and varying pH can have combined effects on the fate of iron oxide nanoparticles
with increasing pH resulting in a higher level of aggregation.
Likewise, surface modification of nanomaterials can influence the environmental fate andbehaviour. As carbon nanotubes are considered to be highly hydrophobic and with atendency to aggregrate, they would be expected to settle in the natural environment.
However, Kennedy et al. (2008) have indicated that surface modifications, which arewidespread (e.g. functional groupings and coatings) lead to increased dispersability,
increased water column stability and lower settling rate, especially in combination withnatural organic matter.
To understand the fate of nanomaterial dispersions in the environment it will benecessary to characterise the nanomaterial colloidal properties and the aqueous phase
physical-chemical properties to a greater degree than is necessary for gas phase ordissolved substances (e.g. present as environmental contaminants). For example,
moderate changes in the ionic strength will have little effect on the solubility or manyorganic substances (e.g. PAHs, most pesticides) but can have major effects on thesuspension stability of nanoparticles. In saline environments nanomaterials have atendency to aggregate (Nielsen et al. 2008, Stolpe and Hassellöv, 2007) and thus would
most likely tend to settle. In sedimentary systems it would be important to determine
how these nanomaterials might interact with organic matter and potentially be adsorbed
and sequestered. This would have influence on their bioavailability and determine their
biological uptake.
The estimation of concentrations in water is essential to environmental risk assessment.
In sharp contrast with the situation for conventional chemical substances, there is neither
theoretical nor empirical evidence that can be used to predict residual concentrations ofnanomaterials in suspension under conditions of limited colloid stability. Whensubstances enter the environment, they distribute themselves between the various
phases of the system (partitioning). The environmental distribution of substances is often
predicted by the octanol-water partition coefficient (Kow). However, there is no reason toassume that the Kow of the substance of which the nanoparticles are made of is predictiveof the extent to which nanoparticles associate themselves with other particles. The Kow is
probably not applicable to non-soluble nanomaterials for risk assessment purposes. Dueto the interactions of nanomaterials with various components of the environmental
35

Risk Assessment of Products of Nanotechnologies
system, generally near-zero concentrations of the nanomaterial in its original form would
be expected. It is of great importance to gain understanding of the environmental
conditions under which stable colloidal suspensions of dispersed nanoparticles can beformed.
For nanomaterials which may be solubilised, Kow could be applicable. Recently, Jafvert
and Kulkarni (2008) have studied the octanol-water partition coefficient (log Kow) of
buckminsterfullerene (C60) and its aqueous solubility. The authors obtained a value forlog Kow of 6.7, and a value for the solubility of C60 in water-saturated octanol of 8 ng/L.
Based upon this high Kow, it is expected that C60 has high affinity for lipids and organic
matter, indicating that in the natural environment, C60 will tend to sorb to solid phases.
Some recent predictive modelling work has been published for TiO2 and silver
nanoparticles and carbon nanotubes (Boxall et al. 2007, Mueller and Nowack 2008).
However current knowledge of the behaviour of nanoparticles in natural waters provides
insufficient basis for the full assessment of environmental exposure concentrations ofdispersed nanomaterials. There is an urgent need to improve knowledge in this area.
It should be noted that in wastewater treatment plants partitioning of nanomaterials into
the solid biomass is likely to be an important fate for hydrophobic materials which end up
in the sewage stream.
Soil and sediments
As described above, depending on receiving environment and material, nanomaterials, if
not degraded or dissolved, will tend to aggregate and eventually settle onto thesubstrate. Within soil and sedimentary systems it is expected that these materials will
adhere to solids.
3.6.1.2. Test methods for predicting environmental distribution
It is likely that the OECD test methods for a number of physico-chemical methods forenvironmental distribution are applicable, although this needs to be further assessed,
taking into account the administration of the sample to the test system.
Methods for assessing the environmental distribution of nanomaterials have been
described (Christian et al. 2008, Hassellöv et al. 2008, Klaine et al. 2008, Tiede et al.
2008;). They are progressively being developed so that the complex issues of fate indifferent media may be addressed. Nevertheless, much information is still needed in thisarea.
For reasons explained above, it is doubtful whether standard tests of vapor pressure,water solubility, octanol-water partition coefficient and ready biodegradability areadequate and sufficient to describe and predict the distribution of nanomaterials in theaquatic environment.
Vapor pressure and solubility
Vapor pressure and water solubility of conventional chemicals are used to predict
air/water partitioning. If measurable at all for nanomaterials, these properties may not
be very useful in predicting the extent to which nanoparticles may partition from water
into air. However, solubility of nanomaterials in water, or rather: the rate of dissolutionof nanoparticles in water is important for an entirely different reason. Toxic effects of thepresence of nanomaterials may well result, at least in part, from the presence ofdissolved species that originate from dissolution of the nanomaterials. To date, it isunclear to what extent the effects observed can be attributed to the dissolved form or to
the nanoformulation the effect being a combination of fraction and size (Franklin et al.
2007, Navarro et al. 2008b). Nevertheless, Lin and Xing (2008) have suggested thatphytoxicity observed on exposures to ZnO nanoparticles may not be attributed solely todissolved zinc. Griffit et al. (2008) drew a similar conclusion with respect to nanosilver.

36

Risk Assessment of Products of Nanotechnologies
The OECD assay on water solubility (OECD 1995) may not be very useful in this context(OECD, 1995). Rather, standard measurement of rate and extent of dissolution undernatural water conditions would be helpful. Many nano materials are highly insoluble inwater, so that specialised methods are likely to be needed to measure or estimate theirwater solubility anyhow. For example, the solubility of fullerene is usually estimated bymeasuring solubility in alcohols and extrapolating to a zero carbon alcohol, i.e. water(Jafvert and Kulkarni 2008).
3.6.1.3. Test methods for degradation and transformation
Environmental persistence of nanomaterials (i.e. resistance to transformation anddegradation) depends on the chemical composition of both core and surface material.
Although it is possible that most nanomaterials will be persistent in their original
particulate form, this cannot be assumed in general. It seems likely that the organic
coatings of nanomaterials are readily transformed or degraded, but there is lack of datain this area. As mentioned above, dissolution may occur for at least some metal
nanomaterials (Franklin et al. 2007, Luoma 2008). Whether or not followed bydegradation of the dissolved material, the process of dissolution makes nanoparticles
disappear and become less persistent.
In standard tests for ready biodegradability of chemical substances, either disappearanceof dissolved organic carbon or the generation of CO2 is measured. Therefore, it isnecessary to examine first whether the nanomaterial can be utilised as an energy or
nutrient source for microorganisms. Secondly, the nanomaterial must be available tomicroorganisms in order to be degradable. If the material is unlikely to reside in thewater column or if it is not soluble in water, biodegradation is unlikely and testing insurface water may be unnecessary.
For C-containing nanomaterials, the biodegradation screening methods (e.g. for readybiodegradability) measuring dissolved carbon are not applicable. In principle, themethods measuring carbon dioxide production or oxygen uptake are applicable, but theyrequire large amounts of test material. It is also important to consider whether carbon
based nanomaterials such as fullerenes and nanotubes can be degraded at all under any
conditions. However, some data indicated that fullerenes could be taken up by wood
decay fungi, suggesting that the carbon from fullerenes could be metabolised (Filley etal. 2005).
Simulation tests for biological degradation in various environmental compartments areapplicable in principle, but again the detection of the nanomaterials is the challenge. The
possible degradation to carbon dioxide, integration into biomass or other partitioning canbe followed using labelled test material. However, it should be noted that the use oflabels needs specific attention in terms of association of the label with the nanomaterial.
Likewise, for hydrolysis testing, the chemical structure of the material and whether it
contains groups which could be subject to hydrolysis dictate whether this test isnecessary or appropriate. In view of the sometimes very long lifetime of ecosystemprocesses, other non biological degradation mechanisms have to be investigated (e.g. UV
induced, slow dissolution).
3.6.1.4. Test methods for bioaccumulation
Current work assessing uptake has focussed on exposures in media with different
nanomaterial loads over a specific time interval, followed by total body burden
assessment, especially if species are small, such as Daphnia species, copepods or
Lumbriculus (Fernandes et al. 2007, Petersen et al. 2008, Roberts et al. 2007). If
organisms are larger, specific studies have focussed on detection, following exposures, ofloads within specific organs, such as liver, kidney, muscle, gills (Handy et al. 2008a). In
terms of detection, it may not always be possible to identify the form of such material.
This may be particularly important for materials that may tend to get into solution suchas silver (Luoma 2008, Navarro et al. 2008b).
37

Risk Assessment of Products of Nanotechnologies
From studies of biological exposures of nanomaterials it is clear that adsorption andaggregation of the material onto surface of the organism is commonly observed(Fernandes et al. 2007, Handy and Eddy 2004, Nielsen et al. 2008, Rosenkranz et al.
2009). This has also been shown by the aggregation of single wall carbon nanotubes on
the gill mucus of rainbow trout (Smith et al. 2007) and of carbon black and titanium
dioxide nanoparticles on the carapaces of Daphnia, (Fernandes et al. 2007), as well as
the entanglement of macroalgae gametes by clusters of carbon black (Nielsen et al.
2008).
Given the tendency of nanomaterials to aggregate, and thus their likelihood to end up
associated with sediment (Klaine et al. 2008) bioaccumulation studies on sedimentorganisms would be especially important. OECD has recently adopted a new standard
test for the assessment of bioaccumulation into sediment worms using Lumbriculus
variegatus. This method could be relevant to be used in a test battery for risk
assessment as OECD has also published recently a toxicity test (OECD TG 225) based on
the same species which could provide effects data (OECD 2008b).
For organic substances, there is an established relationship between octanol/waterpartition coefficient (Kow) and bioaccumulation or bioconcentration factor (BCF). However,this relationship may not hold true for nanomaterials.
The main challenge in testing the bioaccumulation of nanoparticles is their detection andcharacterisation in tissues and body fluids. Radiolabelling could make detection andquantification easy but it has also limitations; for example, the labelled material canbehave differently from the non-labelled particles. Petersen et al. (2008a) used radiolabelled CNTs to assess uptake and depuration by Lumbriculus variegatus. Anotherpossibility could be the radio-activation of metal and metal oxide nanoparticles (Oughton
et al. 2008). It enables both localisation and quantification within tissues or organisms.
This technique is still at experimental stage and a key aspect is how ionisation of
manufactured nanomaterials may interfere with the exposure assay and any results.
Standard BCF testing protocols such as OECD 305 (OECD 1996) may have limitations intesting of bioaccumulation of nanoparticles. It has been observed for substancesdissolved in water that a large molecular size effectively (MW > 600, or effectively adiameter size > 0.5 nm) limits direct uptake. It is likely that in most cases the relatively
large size (1-100 nm) of nanoparticles compared to dissolved molecules limits their direct
uptake by fish gills. Fish dietary bioaccumulation factor (BAF) testing (Fisk et al. 1998;
Stapleton et al. 2004) is not a standard OECD testing protocol yet. This spiked foodmethod is suitable for testing of poorly soluble, large molecules and might be suitable fortesting several classes of nanoparticles, either by itself or in combination with the OECD
305 testing. However, more data using a harmonised OECD dietary protocol, especiallyfor testing nanomaterials, are needed. The testing results of human health endpoints
should also be taken into consideration if available when generating environmental
testing plans for specific nanomaterials. Uptake studies from mammalian studies may
give valuable basic information on uptake characteristics, rates and mechanisms ofnanoparticles also in non-mammalian species.

3.6.2.

Bioavailability and exposure

3.6.2.1. General Principles
Uptake by biota is likely to be via the respiratory or digestive tracts in animals, or via theroot system in plants. Uptake across epithelial surfaces is also possible. Plants and fungi
have cell walls that act as an initial barrier to the entrance of nanomaterials.
In terrestrial (and water) systems some nanomaterials may preferentially bind to NOM(see section 4.6.1.1) and thus become less bioavailable. Sediment feeders may be able
to uptake these nanomaterials. In fact they may preferentially ingest them if they are38

Risk Assessment of Products of Nanotechnologies
associated with NOM (Roberts et al. 2007) and strip/de-associate them within the gastrointestinal tract. Li et al (2008) have reported the reduced bioavailability, and resulting
reduced antibacterial activity, associated with the increased sorption of C60 to soil organic
matter.
In aquatic systems stabilisation by NOM may maintain nanomaterials within the watercolumn which may result in increased bioavailability to aquatic organisms (Kennedy et al.
2008). Although such association with NOM also may reduce and even eliminate
antibacterial activity (Li et al. 2008).
If the nanomaterial readily dissolves in water current protocols and guidelines developed
to measure bioavailability of conventional chemicals are applicable, although in suchcases the test would address the dissolved form rather than the nanoparticulate form.
However, given that depending on the nanomaterial and the receiving environment therate of dissolution could be quite variable, there is a possibility that a combination of
nanoparticles (i.e. size) and substances (dissolved nanomaterial) elicits the detected
toxic effects (Luoma 2008). Limbach et al. (2007) have shown that for partially soluble
nanomaterials such as cobalt oxide and manganese oxide the nanoparticles may be taken
up into cells preferentially to their respective ionic forms. It is then possible that once
inside the cells these nanoparticles may dissolve, resulting in enhanced toxic effects.
Regardless of the key causes, it should be considered that given the increased use, andthus release, of nanomaterials with different levels of solubility this will lead into potential
increased levels of soluble substances which may result in undesirable environmental
effects. These effects may be enhanced (depending on material, receiving environmentand species) by a combination of forms, i.e. particulate and soluble.
For nanomaterials that do not dissolve readily, it should be determined whether they will
form stable dispersions in air or stable suspensions in aqueous media (in both fresh andsea waters).
3.6.2.2. Exposure to nanomaterials in experimental studies
One of the major problems in aquatic ecotoxicological fate and effects testing is theabsence of consistent and broadly-applicable information on how nanomaterials are
suspended in various exposure media used in ecotoxicological testing. There are
essentially three approaches to achieve as uniform as possible stock solutions for testing:dispersion with strong solvents and detergents, dispersion by sonication or dispersion byprolonged stirring (Crane et al. 2008, Klaine et al. 2008). Suspension methods used todate include the use of strong solvents e.g. tetrahydrofuran (THF) (Oberdörster 2004),
dispersion agents e.g. sodium dodecyl sulphate (SDS) (Smith et al. 2007), bath or
ultrasonication with filtration to remove aggregates (Lyon et al. 2006), stirring (HundRinke and Simon 2006, Oberdörster et al. 2006), and combinations of these methods.
Natural organic matter (NOM) can be a suitable dispersant and can keep thenanoparticles suspended longer than a 1% solution of SDS (Hyung et al. 2007). In
addition, the time used during mixing (sonication or stirring) is also very variable (Klaine
et al. 2008). Henry et al. (2007) has referred to some of the key issues regarding theuse of solvents when dispersing nanomaterials in aqueous media.
In most cases, the verification of exposure and the characterisation of the nanomaterials
in the resulting suspensions is limited to working on stock solutions rather than on theactual concentrations, either after a dilution series is generated or periodically over theduration of an exposure or media-renewal period. As such, it is likely that the methods
reported might not produce similar results for different forms of a nanomaterial. In
addition, similar to the testing for health effects also the possible temporal evolution of
nanomaterials during the perfomance of the assays should be considered.

39

Risk Assessment of Products of Nanotechnologies
3.6.2.3. Food chain effects and secondary poisoning
Not much work has been published on potential food chain effects of nanomaterials. A
recent study (Holbrook et al. 2008) on the possible transfer of quantum dots in a
simplified aquatic food chain has found that these materials can be transferred to rotifers
through dietary uptake of ciliated protozoans. Although there was transfer across theselevels, bioconcentration (accumulation from surrounding environment) in the ciliates waslimited and no biomagnification (enrichment across levels) in the rotifers detected. This
study indicates potential for transfer across food chain levels but this would depend on
material type and food chain, as is mostly the case for other studies of standard
materials. Fortner et al. (2005) have also observed that fullerene nanoparticles
accumulate in microbial cells, in worms eating those microbes and possibly in animals
higher up the food chain. Furthermore, Bouldin et al (2008) have reported the transfer ofquantum dots from dosed algae (Pseudokirchneriella subcapitata) to Ceriodaphnia dubia.
Petersen et al. (2008b) have also indicated that CNTs were not readily bioaccumulatedby the earthworm Eisenia foetida with results indicating bioaccumulation factors 2 ordersof magnitude smaller than those measured for pyrene used for comparison.

3.6.3.

Environmental effects

Ecotoxicological testing in soil and sedimentary systems has been the focus of relatively
few studies. As a result, methodology and practical approaches have not been as widely
discussed in the literature. Suspensions of nanomaterials in aquatic media, followed byeither mixing or spraying on sediments/soils would lead to similar issues to the ones
raised above. Other methods include mixing or applying nanomaterials directly to soils
and sediments. It is clear that similar issues of standardisation also apply to thesesystems. There is a need, therefore, to address methodological variability associated withthe current studies assessing the hazard of nanomaterials in environmental models.
Although it is accepted that methods need to be appropriate to the materials being
tested, as well as the test organisms and end points studied, it is important thatstandardised methology is developed and implemented so that variability can be kept toa miminum and results widely accepted and replicated.
In general, it is necessary to check that the suspensions used for aquatic testing aresuitable for the test organisms. Salt concentration, pH, solvent and amount of solvent
have to be within ranges tolerated by the test organisms. For organic and inorganic
nanomaterials different procedures are usually applied. Inorganic nanomaterials (e.g.
metals and metal oxides) are weighed into an aqueous solution; they are homogenously
dispersed by ultrasound or by stirring and followed by filtration. Organic, water-insoluble
nanomaterials are dissolved in solvents such as tetrahydrofuran (THF), toluene or
benzene, or dispersed by detergents. By adding water and removing the solvent, a stableaqueous suspension is obtained. However, there are indications that traces of THF used
to solubilise fullerenes may remain in the suspension resulting in toxicity due to thesolvent (Zhu et al. 2006).
3.6.3.1. Environmental test systems
Microbial systems
Uptake of nanomaterials by microbial organisms might be via diffusion, specific or nonspecific uptake, or via membrane damage (Klaine et al. 2008). The bioavailability andantibacterial activity of C60 fullerene in soil and water were found to be affected by theconcentration of humic acids (Li et al. 2008). In general, the higher the carbon content of
the soil, the stronger the likely adherence to soil/sediment. A list of some microbial
effects observed is presented in Table 1.

40

Risk Assessment of Products of Nanotechnologies
Table 1 Effects of nanomaterials on microbial species (from Klaine et al. 2008,
Wiesner 2006, and references therein)
Nanomaterial

Observed Effects

Carbon-containing
Fullerenes

Antibacterial to a broad range of bacteria, inhibit growthof E. coli by interfering with energy metabolism
Cleave plasmid DNA
Induce DNA damage in plasmids

Carbon nanotubes

Antibacterial to E. coli, cell membrane damage
Cytotoxic to microbes

Metallic
Quantum dots, Silver

Bactericidal; viricidal
May penetrate cells by oxidative damage to membraneGold

Low toxicity to E. coli and Staphylococcus aureus

Metal oxides
TiO2 , MgO, CeO2 ZnOGeneralised anti-bacterial effect

OthersSiO2

Mild toxicity due to ROS production

Generalised microbial effects reported have been: disruption of membrane/membranepotential, production of ROS, oxidation/damage to proteins, interference with electrontransport/respiration, potential DNA damage, with in general more serious effects
observed on Gram-positive species (Klaine et al. 2008 and references therein).
Terrestrial systems
Few studies on the effects of nanomaterials on soil organisms have been published todate. One of the first studied the effects of aluminium oxide nanoparticles on theemergence and growth of plants (Yang and Watts 2005). The authors observed clear
effects but it was later debated whether these effects were due to the nanoparticle formof aluminium or to a soluble fraction of aluminium ions (Murashov 2006). Regardless of
the key cause, it is clear that a negative effect was observed that resulted from theexposure to aluminium in a nanoparticulate form.
Recently, Cañas et al. (2008) studied the effects of single walled carbon nanotubes on
the root elongation of crop species. A few minor effects were detected on some species
after rather short exposure times of 24 and 48 hours. However no uptake of single walledcarbon nanotubes was observed. Lee et al. (2008), using a plant agar test system,
indicated a toxic effect of copper nanoparticles as demonstrated by a reduced growth ofseedlings of mung bean (Phaseolus radiatus) and wheat (Triticum aestivum). Using
transmission electron microscopy and energy dispersive spectroscopy, the authors
observed the accumulation of copper particles in the cells.
Reduced enzyme activities for catalase (CAT) and glutathione-S-transferase (GST) wereobserved after ingestion of titanium dioxide nanoparticles (anatase) by terrestrial isopods(Porcellio scaber) (Jemec et al. 2008). However the overall endpoints like survival andgrowth were not affected. Scott-Fordsmand et al. (2008) detected effects on the41

Risk Assessment of Products of Nanotechnologies
reproduction of earthworms (Eisenia veneta) when the worms were exposed to doublewalled carbon nanotubes in food.
Aquatic systems
Studies focussing on the hazard of nanomaterials to a range of aquatic species werereviewed by SCENIHR (2006). Further developments took place through 2008 with newdata and knowledge generated. Most of the recent studies have indicated that key
aspects of aggregation may result in exposures not reflecting the highest doses. Issues
considering solvents and the role of NOM, as a stabiliser of nanomaterials, have also
been highlighted (see above). Nevertheless, it is important to note that increased
dispersion has not led to increased bioavailability and hazard in all studies. Results
depend on nanomaterial composition (Franklin et al. 2007, Navarro et al. 2008a).
Exposure to raw carbon nanotubes resulted in the viability of Daphnia magna being
reduced, but this was not found in stable dispersions when compared with functionalised
and stabilised forms of carbon nanotubes (Kennedy et al. 2008). In contrast, Kang et al.
(2008) indicated that some methods used to stabilise the dispersion of CNTs (e.g.
functionalisation) resulted in increased toxicity for the bacterial systems they werestudying.
Recent results have indicated the physical interference (e.g. movement hindrance,
clogging) of nanomaterials with biota (e.g. Nielsen et al. 2008). Sublethal effects
observed included lipid peroxidation, altered haematology, changes in behaviour withsome effects being observed at different stages in the life cycle (Zhu et al. 2007).
Interactions of nanomaterials with other pollutants
It has been suggested that nanomaterials may interact with contaminants which may
result in toxic effects on biota (Cheng et al. 2007, Xia et al. 2004). Baun et al. (2008)
indicated the potential of nanomaterials to enhance the toxic effects of organic
contaminants.
3.6.3.2. Methods of assessment in vitro
Methods of assessment in vitro have mirrored work in the area of mammalian toxicology.
For example, several approaches used to study oxidative assessment have been used,
and more slowly work is taking place in the area of genomics and proteomics. Other
methods, borrowed from mammalian work, such as assessment of effects ofnanomaterials on fish hepatocyte function evaluated by enzyme lactate dehydrogenase(LDH) as a marker for cell toxicity (Bopp and Lettieri 2008) or assessment of lysosome
stability in mollusc hemocyte cells, may also be used (Castro et al. 2004).
Linking effects at different levels of organisation is important in the assessment ofpotential long term effects, as well as to improve knowledge on how some toxic effects
may occur. In vivo exposures, followed by assessment of specific endpoints at organ andorganelle levels, as well as biochemical endpoints, provide a comprehensive approach toassessment of effects. In vitro studies, such as the ones described above, allow a focused
assessment of mechanistic effects at a specific level of organisation.
3.6.3.3. Methods of assessment in vivo
There is still much debate in the literature (Fernandes et al. 2007, Handy et al. 2008b,
Klaine et al. 2008) regarding what may be considered optimal approaches for exposures.
Exposure media, mixing or suspension of materials within the media and consideration ofrealistic exposures, have all been a particular focus of attention. In this context, an
important point of consideration has been the characterisation of the nanomaterials in
the exposure studies. This has been particularly debated in studies of sedimentary
systems. Mixing of nanomaterials with sediments/soils, as well as characterisation overtime, are still areas at a very early stage of development.

42

Risk Assessment of Products of Nanotechnologies
In this context, consideration of detection, in a background of natural abundance ofspecific types of materials such as carbon-based products, zinc and silicon, is an areathat is still currently advancing in technical terms.
A wide range of methods have been used to assess the hazard of nanomaterials on
environmental species. The approaches were chosen according to the species studied.
Laboratory studies have focussed on the effects of a range of nanomaterials on standard
species used in ecotoxicology. Most have focussed on aquatic species including: primary
producers (mainly Pseudokirchneriella subcapitata (Franklin et al. 2007, Van Hoecke etal. 2008) and Desmodesmus subspicatus (Hund-Rinke and Simon 2006); invertebrates,
mainly Daphnia species but also other crustacean (Fernandes et al. 2007, Hund-Rinke
and Simon 2006, Lovern and Klaper 2006, Rosenkranz et al. 2009), and fish (such as
rainbow trout Oncorhynchus mykiss, zebra fish Danio rerio, largemouth bass Micropterus
salmoides, fathead minnow Pimephales promelas and Japanese medaka Oryzias latipes;(
Federici et al. 2007, Griffit et al. 2007, Lee et al. 2007, Oberdörster 2004, Smith et al,
2007, Warheit et al. 2007, Zhang et al. 2007). The toxicity to various microbialorganisms has also been studied (e.g. Lyon et al. 2006, Lyon and Alvarez 2008, Sondi
and Salopek-Sondi 2004). In some of these studies, toxic effects were observed.
The integration of endpoints such as mortality, growth, feeding and reproduction, arewidely used in ecotoxicology. In addition, specific biomarkers, such as means ofassessing oxidative stress (in a specific organ, or whole body; e.g. lipid peroxidation),
genetic damage, CYT P450 levels, gene expression, damage to specific cell organelles(e.g. mitochondria or nucleus) are all widely used in the assessment of effects ofnanomaterials. Cytological responses such as cellular apoptosis and necrosis have also
been used. Although the methods employed in these studies tend to be standard
methods used routinely in ecotoxicology studies, modifications have been implemented
to address the specific particle issue, or to address the effects of interference of thematerials with reading of results.
Less research has taken place using soil or sedimentary species but work is now
progressing at a steady pace in these systems. The procedures that were adopted havein general followed OECD guidelines, particularly when using standard species, but also
when other species were used. Depending on the aims of the study, different durations of
exposure have been used.
The number of scientific studies assessing the environmental effects of nanomaterials has
increased dramatically in the period 2007-2008. The main focus is still on microorganisms and invertebrates, followed closely by studies on fish species. Still very muchlacking are studies on soil systems and terrestrial species in general, including primary
producers. There is also a general paucity of studies on marine species. This is not
surprising given the complexity associated with dispersing and suspending nanomaterials
in exposure media. Nevertheless, published results to date indicate clearly the potential
for hazardous effects, at lethal and sublethal levels, including behaviour, reproduction,
growth and development, ROS production, induction of inflammatory responses andcytotoxic effects. In addition, a small number of studies have indicated the potential fortransfer to embryos, accumulation and potential food chain transfer.
Nevertheless, the exposure levels organisms may endure in their natural environments
and how the results in the laboratory can be extrapolated to assess hazard in the field isless clear. Information on environmental loads is at present lacking. One important
aspect in this context is the understanding of any interactions of nanomaterials withmicro-organisms in sewage treatment plants, and the consequent effects on thetreatment process.
A few key issues need to be brought out when assessing critically the results obtained todate on the environmental hazard of nanomaterials in order to focus on what is
important and optimise the approach and design of future studies.
A first issue, which has already been widely discussed, concerns the protocols used inlaboratory exposures and a related link to the current lack of standardised protocols. The
43

Risk Assessment of Products of Nanotechnologies
use of mechanical or chemical means to suspend nanomaterials may lead to changes in
the physical-chemical properties of the test material. It is unclear what the extent ofthese may be and how they may impact any effects observed.
Arguably, dispersants/surfactants/solvents may need to be used in certain situations;however, it is important that they must not add to the toxicity of studied materials. It issuggested that results of studies where THF was used should be treated with caution, as
at least in one study, the observed toxicity was due to traces of THF (Zhu et al. 2006).
The same caution may apply to other dispersants for which there is lack of knowledge
regarding their interaction with the test material (e.g. SDS). Further work with humic
and fulvic acids, as well as widely used detergents (which are likely to be encountered in
the environment) should be undertaken.
Related to this topic is the use in hazard assessment of ready-made (off-the-shelf)suspensions of nanomaterials. It is unclear what the interactions might be of the usedpreparation dispersants on the properties (and thus behaviour) of the test material (asdescribed above). Thus any reported effects might not be comparable with effects
observed on exposures of the same species to the same component material but which isin a different form (i.e. solid and suspended nanomaterials in the laboratory vsnanomaterials obtained as a suspension).
Therefore, standardisation of protocols, as possible, is desirable for the comparability ofstudies as well as reliability of results, and the derivation of information to risk
assessment and risk management.
Regarding experimental design and approach, characterisation of exposures, viaappropriate method(s) should be carried out and chemical analyses undertaken, as
possible. The assessment of the solubility of the nanomaterials being studied is very
important in this context so that any observed effects can be attributed to the different
fractions. This is particularly important in the case of certain metal nanomaterials, as wellas in the case of CNTs and quantum dots.
Some studies have highlighted the importance of assessing contamination of thenanomaterials being studied. This should be undertaken for similar reasons. Anotherimportant point is the comparison of effects between nano and equivalent, larger,
material. This has not been consistently incorporated in the published studies and would
also allow the correct attribution of effects.
There is a lack of information regarding the fate and form of the test nanomaterials
within biological systems following in-vivo exposures. It is unclear what particular form
(e.g. soluble or particulate) is preferentially taken up into tissues and cells. It is likelythat this would depend on the material composition; nevertheless these studies are notroutinely carried out.
Studies should be conducted on a range of guilds and endpoints, with fate within the
body and tissues assessed and depuration quantified, as possible. Micro/mesocosms
studies should be undertaken. Furthermore, dietary studies, the role of nanomaterials’
coatings in uptake and translocation within the body, should be conducted, as well as theassessment of the role, if any, of their interaction with other environmental
contaminants.
In this context it is crucial to ascertain the fate of nanomaterials in the environment sothat their availability for environmental exposure can be assessed. Environmental fate
and load assessment of nanomaterials must, therefore, be undertaken. The use of thecurrent approach to the derivation of Kow in the assessment of environmental fate isunlikely to be beneficial to risk assessment. Nevertheless, the derivation of alternative
approaches may be useful and may allow the development of appropriate predictivemodelling. Finally, further information on the degradability (bio and abiotic) of
nanomaterials should be derived.

44

Risk Assessment of Products of Nanotechnologies
3.7. NANOTECHNOLOGIES- RISK ASSESSMENT
A suitable framework for the assessment of all engineered nanomaterials requires
exposure and hazard data on a wide range of products. At present there have been
insufficient published studies to establish a detailed framework. Nonetheless in theprevious SCENIHR opinion an outline for such a framework, in the form of an algorithm,
was presented (SCENIHR 2007a). This framework remains appropriate although a few
further details can be added in the light of recent publications.

3.7.1.

Relevant physicochemical properties

The most important properties of a nanomaterial to characterise, from a risk assessment
viewpoint, are:
•
•
•
•
•

Size and size distribution of free particles and fibres/rods/tubes. These may be
produced during the manufacture, use (including wear) and/or disposal/recyclingof the nanoproduct.
Specific surface area
Stability in relevant media (including the ability to aggregate and disaggregate)
Surface adsorption properties
Water solubility

In addition, suitable measurements of chemical reactivity are needed although at present
the most relevant ones for a particular nanoparticle/nanofibre are best judged on a caseby case basis bearing in mind the likely applications of the product (see read across).
Depending on the nature of the nanoparticle/nanofibre it may also be appropriate toconsider:••Photoactivation. Recent data have indicated that some nanoparticles may, by
virtue of their relatively large surface area and reactive potential, become
activated by light. This is relevant both in considering their stability and theirpotential to be photo activated when in contact with the skin/external surfaces ofother species.
Potential to generate active oxygen. Production of active oxygen is one accepted
general mechanism for the adverse effects of nanoparticle/nanofibre. Thus the invitro measurement of the ability of a particular nanoparticle/nanofibre to generate
active oxygen species may be considered.

3.7.2.

Read-across

There is insufficient information to identify opportunities for read-across based on thegeneral chemical composition of nanomaterials. Nonetheless there are some properties
for which read-across is appropriate in determining the experimental studies that need tobe considered.
i)ii)Fibres, rods and tubes. In the light of experience with asbestos, and the recent
studies on carbon nanotubes (reported above) if there is a potential exposure
to free fibres, rods and tubes that are chemically/ biologically persistent, arerigid and have a high aspect ratio (i.e. micrometres in length and nanometresin diameter) the possibility should be considered that they may have similar
properties to asbestos.
Particles. There is a large amount of data on airborne fine particles generated
as a result of combustion to indicate that comparable particles may cause
respiratory and cardiovascular effects. Situations in which there is productionof fine nanoparticle/nanofibre with reactive surfaces could cause comparable
effects.

45

Risk Assessment of Products of Nanotechnologies
iii)

iv)

Nanomaterials of comparable dimensions and surface properties.The databasefor extrapolation is very limited, however, for the assessment of a specificnanomaterial it may be possible to highlight relevant properties that requireparticular assessment.
Bulk material. For any nanomaterial for which significant exposure of man orother species could occur, it is appropriate to consider the toxicological/
ecotoxicological properties of the material in other physical forms, unless thereis good evidence that no bulk material will be released in biological systems.

3.7.3.

Development of the risk assessment framework

3.7.3.1. Development of the SCENIHR algorithm
In the previous opinion of the SCENIHR (2007a) a four tier algorithm was presented inwhich the initial consideration was the potential for exposure of man and/or otherenvironmental species to the nanomaterial. There are no new data that would suggest asignificant change in the SCENIHR exposure driven framework to be appropriate, otherthan the aspects discussed above.
This four stage algorithm offered a framework for the case by case evaluation of thepotential risks due to exposure of humans and other species to nanomaterials. The
algorithm is exposure–driven. To be of practical value a thorough assessment of thepotential exposure of humans and other environmental species during the entire life cycle
is vital. This must include not only the current use but also possible further applications.
In addition, it must take into account the potential for nanomaterials to be released
during use (e.g. as a consequence of wear and tear), and the range of end-use fates,that may occur (e.g. waste disposal or waste recycling options).
It is anticipated that as the scientific knowledge improves, it may be possible to classify
nanomaterials into specific risk categories that might become the subject of category
specific risk assessments. However, at present this categorisation is not possible. New
significant developments have come to light (as identified above), which allows further
work on Stage III (Hazard identification and characterisation). These include:
i)
ii)
iii)
iv)

Cell/tissue uptake tests;
Bioaccumulation tests to assess prolonged exposure;
Selection of a test system(s) for nanofibers/tubes that are biopersistent, arerigid and have a high aspect ratio (HAR – L>20µm);
Ability of nanomaterials to trigger one or more of the putative mechanisms of
toxicity (e.g. generation of reactive oxygen species).
3.7.3.2. Addressing deficiencies in the data baseAs discussed above, one of the major challenges for nanotechnologies (and indeed forother emerging issues) is how to characterise the risks where the database is very
limited.
The traditional approach to address such a situation is to adopt a traditional risk
assessment framework and either:– introduce a default value for each major data gap– compare the new/potential nano product with the conventional (non-nano)product
However, other approaches to the risk assessment of nanomatertials exist. There havebeen several recent reviews of the emerging health issues from products ofnanotechnologies (Hannah and Thompson 2008, Hoyt and Mason 2008, Linkov et al.
2007, Sweet and Strohm 2006, Wardak et al. 2008,). Two approaches for coping withthe large gaps in the data have emerged from these publications:46

Risk Assessment of Products of Nanotechnologies– Application of lifecycle methodology currently used to evaluate sustainability thathas a lesser data requirement.
Sweet and Strohm (2006) have proposed a structured approach that combines risk
assessment and risk management approaches viz:
– Is it likely that the product system contributes to actual harm in the life cycle?
– How much does each product or stage contribute?
– Do relevant toxicity data exist for risk assessment?
– What is the potential upstream and downstream technology units impacted?
– What opportunities are available for upstream or downstream improvements (e.g.
environmental quality, emission reductions)?
– What opportunities to control the risks by selecting less risky options or by
restricting access to the hazard or life cycle stage of concern?
Application of this approach, using worked examples would enable the general utility ofthis approach to be assessed
Von Gleich et al. (2008) have applied life cycle inventory analysis to assess the potential
risks and benefits of several eco-efficient nano surface coatings for metals compared to
the non nano surface coating equivalents. This approach appears to be promising to
identify the general environmental impacts of a nanomaterial in terms of use of resources
including energy, but the value of its application to issues concerning human health is as
yet uncertain.
– Use of expert judgment to fill the critical gaps.
One interesting aspect of this approach is its use to identify measures that should betaken for containment or environmental control in the workplace. The approach uses amatrix to characterise the level of concern and the consequent action that should betaken (See Table 2). It involves a two dimensional matrix of the likelihood of exposureand the likelihood of effects and severity. With modification it could be applied to identify
the potential impacts of individual nanomaterials on human health and on other species
along the following lines:
Table 2: Expert Judgement matrix
EXPOSURE TO NP/NF
probable

likelyLesslikelyExtremely
unlikelyEFv. high

4

4

3

3

F

high

4

4

3

2

E

medium

4

4

2

2

C

low

3

3

1

1

T

v. low2

1

1

1

Where 4 =highest priority for a detailed assessment/ preventative action, 1= the lowest
priority for detailed assessment/regulatory action. This type of methodology has been applied
to air fresheners incorporating nanoproducts and to a range of other consumer products(Wardak et al. 2008) although these authors use graphic plots rather than a matrix format topresent the findings.

47

Risk Assessment of Products of Nanotechnologies

Such a methodology could prove useful as a first step, for example, in achieving
consensus among experts and risk managers on priorities. It may also be of value in amatrix or a graphic form to compare products/exposure situations in a transparent
manner.
Von Gleich et al. (2008) have proposed that the assessment of the potential impacts ofnanomaterials should run closely in parallel with the research and technical work todevelop them. They refer to this process as ‘leitbuilder’. This is a logical concept that is
worthy of further elucidation.
A more structured way of selecting which nanoproducts to be developed has been
proposed by Linkov et al. (2007) using multi-criteria decision analysis. Their approach isthat, prior to comparison of individual nanomaterials; expert judgment is used to set
weighted values for relevant parameters of health and ecological effects (e.g. publichealth effects, effects related to occupational exposure, environmental effects),
importance to society and stakeholder preference. The challenge with such an approachis to set the most appropriate weighting, in a manner that is both transparent andacceptable to all the key stakeholders.
The Swiss Federal Office of Public Health (2008) has proposed a structured scoring
system for the categorisation of risks posed by nanomaterials into two classes A and B.
The approach is compatible with the above approach. The key parameters incorporated
into this system are:
- Exposure of human beings / release into the environment- Potential effects (e.g. stability in biological systems (biopersistence), redox
activity)- Nano-relevance (e.g. physico-chemical properties) and- Information about the life-cycle (e.g. is the future life-cycle known?).
The approach is sound but needs to be evaluated using actual case histories. One
possible concern is the use of a single total numerical score to assign nanomaterials as
either low risk or possible risk.
3.7.4.

Conclusion for the risk assessment

The development of a widely accepted and robust methodology that would be used at the
R&D stages to identify and mitigate potential human health (including occupational
health) and environmental risks, associated with individual nanomaterials should begiven high priority. For this purpose it is vital to develop a data bank of case histories toassess its validity.

3.8. Research needs
The research needs as indentified by SCENIHR in its Opinion on the evaluation of theTechnical Guidance Documents (TGDs) are still valid (SCENIHR 2007a). They include
amongst others the availability of validated in vitro assays, development of an approachfor using quantitative structure activity relationship (QSAR), studies on potential
cardiovascular effects, evaluation of bacterial genotoxicity assays, methodology on
prediction of environmental concentrations (PEC), and environmental species used for
ecotoxicity testing. Some progress has been made in the areas of properties needed todetermine for nanomaterial characterisation, toxicokinetics and genotoxicity testing withmammalian cells, and environmental behaviour of nanomaterials. Recent research has
also identified new concerns in the areas of protein fibrillation, potential hazards of48

Risk Assessment of Products of Nanotechnologies
certain nanotubes, and potential for transfer across the food chain in environmental
species.

3.8.1.

Characterisation of nanomaterials

There is a need for comparable, reproducible and repeatable harmonised methods formeasuring and characterising nanomaterials (SCENIHR 2006), especially for measuringconcentrations and characteristics of nanomaterials in biological and environmental
media. Being able to address these gaps is important for providing meaningful data
which can produce a system of reliable risk assessment. This requires also defining themetrics most appropriate for hazard characterisation and exposure, including themethodology to perform the measurements.
There is an urgent need for the development of reference nanomaterials for theevaluation of both the quality of measurement techniques and to compare biologicalresponses.

3.8.2.

Determination of human exposure

Exposure determinations/estimations at workplace and the ambient environment need
improvement. Therefore more specific measurement methodologies need to become
available to discriminate beween background and manufactured nanomaterials. This canbe achieved by working on the feasibility for routine assessments, development of
reliable measurement techniques, standardisation of measurement techniques,measurement strategies, and implementation of screening/monitoring of nanoscaleparticles in sensitive work areas. Challenges are currently especially seen in the detection
and assessment of nanoparticles from products in the environment.
Exposure estimates from food and consumer products remain difficult. Information on
the presence of manufactured nanomaterials solely relies on information (claims)
provided by manufacturers. In addition, exposure estimation is also hampered by lack of
information on product use and use of multiple products containing manufactured
nanomaterials. Coordinated efforts and research strategies for a comprehensive exposure
assessment of manufactured nanomaterials need to be defined.

3.8.3.

Identification of human hazards

The effect of nanoparticles on protein behaviour as demonstrated in vitro needs further
investigation. It is necessary to elucidate whether the in vitro observed effects on protein
fibrillation processes (both enhancement and retardation) also occur in an in vivo
situation or in more complex biological fluids where competitive binding may take place.
There are indications that after deposition at the olfactory mucosa of the nose, ambient
air and nanoparticles may translocate into the brain. This may offer a potential route ofentry for medicinal products into the brain. This observation may also raise some concern
in view of the amyloid diseases of the brain in the context of the potential of
nanoparticles to cause protein fibrillation in vitro. This is certainly an area for which
additional research is urgently needed.
Additional studies on the potential hazards of nanofibers/nanotubes need to be
performed.

49

Risk Assessment of Products of Nanotechnologies
3.8.4.

Environmental exposure

The estimation of relevant environmental exposure concentrations is seriously hampered
by lack of the two essential pieces of information/knowledge. Firstly, there is noquantitative knowledge on the rates of release of nanomaterials to the environment.
Secondly, there is neither knowledge nor theory that can be used to predict
concentrations of nanomaterials in the ambient environment from release rates. Wellestablished knowledge of distribution and fate of chemical substances, as it is applied in
the current EU guidelines for environmental risk assessment of conventional chemicals,cannot be used for nanomaterials without modification. It is recommended that research
be initiated to develop quantitative theory and models that predict residual
concentrations of free nanoparticles from release rates, and implement such models in
the current EU guidelines for environmental exposure assessment of nanomaterials.
One of the unknown processess relevant to the environmental exposure assessment of
nanomaterials is the extent/rate of dissolution of nanomaterials in water. It is unlikelythat the standard OECD methods for measuring solubility of nanomaterials in water can
provide the required information and it is recommended to revise these methods toaccommodate the measurement of the rate of dissolution of nanomaterials in the naturalenvironment.
Most urgently needed are analytical methods to detect and measure ambientconcentrations of free nanomaterials. Currently, no standard methods exist for thispurpose, although efforts are being developed in this area and environmental exposurelevels are still unknown.

3.8.5.

Environmental hazards

Studies on soil systems and terrestrial species in general, including primary producers
are still lacking. There is also a general paucity of studies on marine species.
One important aspect in this context is the understanding of any interactions ofnanomaterials with micro-organisms in sewage treatment plants, and the consequenteffects on the treatment process.
Further work on the establishment of standard protocols is required. The use of
mechanical or chemical means to suspend nanomaterials may lead to changes in thephysical-chemical properties of the test material. It is unclear what the extent of these
may be and how they may impact any effects observed.
Arguably, dispersants/surfactants/solvents may need to be used in certain situations;however, it is important that they must not add to the toxicity of studied materials. It issuggested that results of studies where THF was used should be treated with caution.
The same caution may apply to other dispersants for which there is lack of knowledge
regarding their interaction with the test material (e.g. SDS). Further work with humic
and fulvic acids, as well as widely used detergents (which are likely to be encountered in
the environment) should be undertaken.
Related to this topic is the use in hazard assessment of ready-made (off-the-shelf)suspensions of nanomaterials. It is not clear how the dispersants used in the preparationof the nanomaterials might interact with the test material, and what effects they may
have on the properties (and thus behaviour) of the test material (as described above).
Thus any reported effects might not be comparable with effects observed on exposuresof the same species to the same component material but which is in a different form (i.e.
solid and suspended nanomaterials in the laboratory vs nanomaterials obtained as asuspension).
Regarding experimental design and approach, characterisation of exposures, viaappropriate method(s) should be carried out and chemical analyses undertaken, as
possible. The assessment of the solubility of the nanomaterials being studied is very50

Risk Assessment of Products of Nanotechnologies
important in this context so that any observed effects can be attributed to the different
fractions. This is particularly important in the case of certain metal nanomaterials, as wellas in the case of CNTs and quantum dots.
The importance of assessing contamination of the nanomaterials has been highlighted.
The comparison of effects between nano and equivalent, larger, material needs to beundertaken. This has not been consistently incorporated in the published studies andwould allow the correct attribution of effects.
There is lack of information regarding the fate and form of the test nanomaterials withinbiological systems following in vivo exposures. It is unclear what particular form (e.g.
soluble or particulate) is preferentially taken up into tissues and cells. It is likely that thiswould depend on the material composition; nevertheless these studies are not routinely
carried out.
Studies should be conducted on a range of guilds and endpoints, with fate within the
body and tissues assessed and depuration quantified, as possible. Micro/mesocosms
studies should be undertaken. Furthermore, dietary studies, the role of nanomaterials’
coatings in uptake and translocation within the body, should be conducted, as well as theassessment of the role, if any, of their interaction with other environmental
contaminants.
In this context it is crucial to ascertain the fate of nanomaterials in the environment sothat their availability for environmental exposure can be assessed. Environmental fate
and load assessment of nanomaterials must, therefore, be undertaken. The use of thecurrent approach to the derivation of Kow in the assessment of environmental fate isunlikely to be beneficial to risk assessment. Nevertheless, the derivation of alternately
approaches may be useful and may allow the development of appropriate predictivemodelling. Finally, further information on the degradability (bio and abiotic) of
nanomaterials should be derived.

51

Risk Assessment of Products of Nanotechnologies
4. OPINION
While risk assessment methodologies for the evaluation of potential risks of substancesand conventional materials to man and the environment are widely used and are
generally applicable to nanomaterials, specific aspects related to nanomaterials stillrequire further development. This will remain so until there is sufficient scientificinformation available to characterise the harmful effects of nanomaterials on humans andthe environment. The methodology for both exposure estimations and hazard
identification needs to be further developed, validated and standardised. The highest
risk, and thus concern, is considered to be associated with the presence or occurrence of
free (non bound) insoluble nanoparticles either in a liquid dispersion or airborne dusts.
Characterization of manufactured nanomaterials
For the characterisation of manufactured nanomaterials, several issues are important. A
consensus is now emerging about what properties need to be determined for risk
assessment purposes. In biological test systems nanomaterials may change theirproperties. In particular, they may partly dissolve or agglomerate/aggregate so that theparticle size distribution changes. For (partially) soluble nanomaterials the toxicity may
be governed at least in part by the soluble species/fraction released from thenanomaterial. For low solubility or a slow release, the particulate nature of the substance
may be relevant with regard to potential tissue distribution and local release of toxic
species which should then be considered in the risk assessment of such nanomaterials.
When using nanomaterials, an extensive characterisation is necessary, including thenanomaterial as produced and the nanomaterials as used in test systems and thenanomaterial as present in final products. The characterisation ‘as manufactured’
provides information for the material safety data sheet (MSDS) of the product itself. The
characterisation ‘as used’ in biological systems is needed as properties of nanomaterials
may considerably change, notably the size distribution due to agglomeration/aggregationof the particles. An issue of specific importance are the properties of the nanomaterial as
it is actually used in products and to which consumers may be exposed. For the risk
assessment the latter characterization is of highest relevance.
Legally, in the EU, nanomaterials are covered by the definition of substance within the
REACH regulation (Regulation (EC) No. 1907/2006) (European Commission 2006).
However, the definition of nanoscale is still under debate. Various organisations haveproposed definitions of nanoscale using an upper limit of about 100nm. It should be
noted that most currently proposed definitions use the size of the primaryparticle/structure as a starting point. However, when a nanomaterial is in particulate
form, the particles may be present either as single particles or as agglomerates oraggregates. Depending on the nanomaterial the majority of the particles may be
agglomerates or aggregates. This may lead to the misinterpretation that agglomerates oraggregates of nanoparticles that have external dimensions well beyond 100nm are notconsidered nanomaterials. Yet they retain specific physicochemical properties which arecharacteristic of nanomaterials, most likely due to their large specific surface area (SSA).
Therefore, when describing a nanomaterial it is important to describe not only the mean
particle size but also the size of the primary particles. In addition, information on thepresence of agglomerates or aggregates should be presented. Besides size, the specificsurface area as determined by the BET method is a good metric to describe particulates
as it is independent of the primary versus the agglomerated state. Hence, it should beconsidered to complement the current definition based on physical size by adding a limitof the specific surface area. Solid spheres of 100 nm with unit density have a specificsurface area of 60 m²/g.
There is currently a need for reference nanomaterials. Some are available but they arespherical model materials which are certified primarily for size and are used mainly tocalibrate instruments which measure particle size. The absence of well-definedparameters to measure and of standardised test protocols is identified as a major
obstacle for reference material production. It should be noted that for use in biological52

Risk Assessment of Products of Nanotechnologies
systems certain compounds need to be added which may have an effect on nanomaterial
composition and properties resulting in changes in (toxic) behaviour.
Human exposure
One of the main limitations in the risk assessment of nanomaterials is the general lack ofhigh quality exposure and dosimetry data both for humans and the environment. One of
the issues is the difficulty to determine the presence on nanomaterials and properly
measure them. In contrast to the situation for other exposure routes, for air-borne
nanomaterials, analytical instruments are generally available to determine exposure (sizedistribution of mass and number). This is particularly true in the context of test
atmospheres. However, differentiation between background and incidental exposure is
generally not possible in real life situations as the methods employed mainly measure thepresence of (ultrafine) particles and do not discriminate between the different types ofparticles that may be present. To date most information on particle measurements comesfrom airborne measurements at the workplace. No quantitative or qualitative
measurements of manufactured nanomaterials in ambient air outside of workplaces havebeen identified. Even when outside measurements are available these may be
confounded, an example being carbon nanotubes that also may originate from general
combustion processes and thus can be found in ambient locations. This illustrates thedifficulty of identifying exposure levels of airborne manufactured nanomaterials. There isa need to establish reliable and standardised measurement techniques, to develop
measurement strategies, and to implement screening/monitoring of nanoscale particles
in sensitive work areas. Challenges are currently especially seen in the detection andassessment of manufactured nanoparticles in the environment. This is even more urgent
for exposure of humans and ecosystems via natural water, sediment and soil.
Exposure estimates for consumers from food and consumer products remains difficult.
Information on the presence of manufactured nanomaterials solely relies on information(claims) provided by manufacturers. In addition, exposure estimation is also hampered
by lack of information on product use and use of multiple products containing
manufactured nanomaterials. In a similar fashion to air measurements, determination of
manufactured nanomaterials in consumer products suffers from the difficulty of
discrimination between background and intentionally added manufactured nanomaterials.
Coordinated efforts and research strategies for a comprehensive exposure assessment of
manufactured nanomaterials still have to be defined. The main issues may besummarised as problems in replicating actual exposure conditions in laboratory tests andthe lack of general availability of robust and specific measurement methods. Exposure
assessment needs to consider each stage in the life-cycle.
Human hazard
When nanomaterials come into contact with a biological fluid they may become coatedwith proteins and other biomolecules. The coating may then influence the outcome of thebiological response to the nanoparticle. Coating proteins have been most widely studiedin mammalian systems. The significance of nanomaterial coating for nanomaterial safety
and nanomaterial risk assessment is clear, as it implies that detailed characterisation of
the nanoparticles in the relevant biological environment is necessary. The coating of
nanoparticles may be used for therapeutic purposes to prolong circulation time (byPEGylation) or to target specific locations (e.g. apolipoprotein E for brain,
immunoglobulins for tumors).
As the protein coating may affect the nanomaterial behaviour including its biologicaleffect, it may be anticipated that nanomaterials may have an effect on protein behaviour.
Some nanoparticles have been found to have the potential to promote and to retard
protein assembly into amyloid fibrils in vitro. These experiments were performed using
an incubation of various nanoparticles with purified β2-microglobulin or β-amyloid
protein. Whether the observed nucleation process also occurs in an in vivo situation or inmore complex biological fluids where competitive binding may take place remains to be53

Risk Assessment of Products of Nanotechnologies
determined.
Existing data show that nanoparticles can enter the circulation from the respiratory tract
or the gastro-intestinal tract but typically in minimal amounts (less than 1% percentageof the dose as expressed in mass units). However, although minimal in percentage thismay result in a systemic availability of a considerable number of nanoparticles.
Nanoparticle migration is likely to depend on the physico-chemical properties of thenanoparticles such as size and on the physiological state of the organs of entry. Whenthe nanoparticles reach the blood circulation, the liver and the spleen are the two majororgans for distribution. Circulation time increases drastically when the nanoparticles arehydrophilic and their surface is positively charged. A coating like polyethylene glycol(PEGylation) also increases the residence time in the circulation. For certain nanoparticles
all organs may be at some risk. For all organs investigated so far, either the chemicalcomponent of the nanoparticles or the nanoparticles themselves could be detected, as
demonstrated for the brain and the testes. In the case of distribution to the foetus inutero, contradicting results were observed. The knowledge on toxicokinetics has been
increased showing that, for a given substance, the smaller nanoparticles do have a muchwider organ distribution than the larger nanoparticles.
There are indications that after deposition at the olfactory mucosa of the nose
nanoparticles may translocate into the brain. This may offer a potential route of entry for
medicinal products into the brain. Because of the potential of nanoparticles to cause
protein fibrillation in vitro, this observation may raise some concern in view of theamyloid diseases of the brain. This is certainly an area for which additional research isurgently needed.
Based on the observations on the effects of particulate matter present in air pollution
some concern exists on the possible effect of manufactured nanoparticles on the
cardiovascular system. However, this has not been clearly demonstrated to be the casefor manufactured nanoparticles so far. Overall the information on the possible hazard ofnanoparticles for cardiovascular effects is rather limited and needs expansion.
When carbon nanotubes have physico-chemical and biopersistence characteristics similar
to those of hazardous asbestos fibres, it was demonstrated that they can induce similar
inflammatory reactions. The main characteristics for this to occur are a long thin fibrousform (length >20 µm), rigidity, and no degradability (biopersistence). Whether inhalation
exposure to such carbon nanotubes would pose a risk to humans is unknown. Thus,
manufacturers of nanotubes (possibly of any chemical composition) should be aware thatcertain characteristics (e.g. length, rigidity, biopersistence) may pose a risk and thepossibility for chronic inflammation and mesothelioma induction and consequently should
be considered in the safety evaluation.
The genotoxic effects of conventional particles are driven by two mechanisms: direct andindirect (mediated by inflammatory processes). Nanoparticles may act via either of thesepathways since they can cause inflammation and can also enter cells and cause oxidative
stress. There is some evidence that the small size may allow nanoparticles to penetrate
into sub-cellular compartments like the mitochondria and nucleus. The presence of
nanomaterials in mitochondria and the nucleus opens the possibility for oxidative stressmediated genotoxicity, and/or direct interaction with DNA. For some manufactured
nanomaterials genotoxic activity has been reported, mainly associated to reactive oxygen
species (ROS) generation, while for others contradicting results were obtained. Besides
oxidative stress, additional mechanisms of genotoxicity which may be specific fornanomaterials also need to be considered, such as possible mechanical interferences
during cell division, and other sources of genotoxic effects (i.e. metal release bynanomaterials).
The main issues for human hazard identification may be summarised as a need to ensurethat each test system is appropriate for nanomaterials and to ensure that endpoints ofpotential particular concern (e.g. cardio-vascular effects) are properly addressed.

54

Risk Assessment of Products of Nanotechnologies
Environmental exposure
The increasing production, use and disposal of nanomaterials will lead to an increase inenvironmental exposure. Similar to human health risks, fate and behaviour of the
manufactured nanomaterials in the environment itself is crucial for the potential ecotoxic
effects in various environmental species. Estimation of relevant exposure concentrations
is seriously hampered by lack of the two most important pieces of
information/knowledge. Firstly, there is no quantitative knowledge on the rates of releaseof nanomaterials to the environment. Secondly, there is hardly any knowledge on theconcentrations of nanomaterials in the ambient environment. There is also no theory thatcan be used to estimate such concentrations from release rates. The main problem isthat the well-established knowledge of distribution and fate of chemical substances, as it
is applied in the current EU guidelines for environmental risk assessment of conventional
chemicals, cannot be used for nanomaterials without modification. Most certainly, the Kow
is of limited use as a predictor of the extent to which nanomaterials adhere to solid
surfaces.
A hypothesis to describe fate and distribution of nanomaterials is slowly being developed,
mostly from classical knowledge of colloid science. It is recognised that the main factorsthat influence the colloidal behaviour of nanoparticles (aggregation/agglomeration,
sedimentation) are, besides the physical and chemical properties of the nanomaterial,
the properties of the receiving environment: pH, ionic strength, prensence of naturalorganic matter. Depending of the nanomaterial characteristics either an increased
sedimentation or improved dispersion of nanomaterials in water may occur. Exposure
estimates are hampered by difficulties in distinguishing manufactured nanomaterials frombackground levels of naturally occurring nanomaterials. For the environmental risk
assessment the estimation of water concentrations is essential. The assessment of
exposure concentrations of dispersed nanomaterials requires detailed insight into theprocesses that act on the particles in the environment. However, currently available
knowledge of these processes is insufficient to allow quantitative predictions of theenvironmental fate of nanomaterials.
The solubility of the nanomaterials is an important property that needs to be addressed.
Knowledge of the extent to which nanomaterials dissolve and the rate at which this takes
place is essential in two respects: (i) it is a direct control of the concentrations ofnanomaterials in the environment and of the time that the nanomaterials reside in theenvironment and in organisms, and (ii) it determines the concentrations of dissolved
species that originate from the nanomaterials. Knowledge of the extent to which
nanomaterials dissolve in water, and of the rate at which this occurs, is essential topredicting the environmental fate and the effects of nanomaterials. It is doubtful whether
currently available standard methods for measuring the (rate of) dissolution canadequately deliver this knowledge.
Unlike in the assessment of exposure concentrations of conventional (dissolved) chemical
substances, the octanol-water partition coefficient Kow is likely to have a limited role inpredicting water-solids partitioning. An alternative theory to predict the exposure levelsof nanomaterials in water is yet to be developed. Based on well-established knowledge of
colloid science, it is expected that pH, ionic strength and presence of natural organic
matter in the water compartment (freshwater versus marine environments) are
important factors influencing the residual levels of nanomaterials in suspension.
Depending on these factors and the chemistry of the manufactured nanomaterial
increased aggregation and thus sedimentation or in contrast enhanced dispersion may
occur.
For some nanomaterials (i.e. quantum dots) the transfer across environmental species
was demonstrated indicating a potential for bioaccumulation in the species at the end ofthat part of the food chain. The main issues may be summarised as the development ofsuitable methods to assess the distribution of nanomaterials in the environment and thelack of portable monitoring equipment to measure levels of nanomaterials in different
environmental media.
55

Risk Assessment of Products of Nanotechnologies
In addition, for many manufactured nanomaterials the methods currently used (carbon
dioxide production, integration into biomass) for determining biological degradation will
not be applicable.
Environmental hazard
Ecotoxic effects on environmental species have been demonstrated; aquatic species havebeen most studied. One of the major problems in ecotoxicological fate and effects testingis the absence of consistent and broadly-applicable information on how nanomaterials areto be suspended in various exposure media used in testing. Exposure media, mixing ofmaterials with the media and consideration of realistic exposures, need a particular focus
of attention. In this context, the characterisation of the nanomaterials in theeco(toxico)logical studies is important. Mixing of nanomaterials with sediments/soils, aswell as characterisation over time, are areas which are still at a very early stage ofdevelopment. In addition, there is the problem of the presence of background levels of
nanomaterials and how to distinguish them from the nanomaterials being tested.
The common endpoints used in ecotoxicology such as mortality, growth, feeding, andreproduction can also be used for the evaluation of ecotoxicity by nanomaterials. In
addition, some biomarkers similar to those used in the assessment of mammalian
toxicity, such as oxidative stress, genetic damage and gene expression, may provide
some insight in toxic mechanisms of nanomaterials.
The main issues for environmental hazard assessment may be summarised as the needfor validation of laboratory test systems for characterising the effects of nanomaterials
and the need for studies of the impacts of specific nanomaterials on ecosystems.
Risk assessment
Health and environmental hazards have been demonstrated for a variety of
manufactured nanomaterials. The identified hazards indicate potential toxic effects ofnanomaterials for man and the environment. However, it should be noted that not all
nanomaterials induce toxic effects. Some manufactured nanomaterials have already beenin use for a long time (e.g. carbon black, TiO2) showing low toxicity. Therefore, thehypothesis that smaller means more reactive, and thus more toxic, cannot be
substantiated by the published data. In this respect nanomaterials are similar to normal
chemicals/substances in that some may be toxic and some may not. As there is not yet agenerally applicable paradigm for nanomaterial hazard identification, a case-by-caseapproach for the risk assessment of nanomaterials is still recommended.

56

Risk Assessment of Products of Nanotechnologies

5. MINORITY OPINION
none

57

Risk Assessment of Products of Nanotechnologies

6. LIST OF ABBREVIATIONSAASAtomic Absorption Spectrophotometry

ADME

Absorption, distribution, metabolism and excretion

ADP

Adenosine diphosphate

BAF

Bioaccumulation factor

BBB

Blood Brain Barrier

BCF

Bioconcentration factor

BTB

Blood Testis Barrier

CAT

Catalase

CB

Carbon black

CEN

European Committee for Standardisation

CNT

Carbon Nanotube

CYT

Cytochrome

DNA

Deoxyribonucleic acid

ECHA

European Chemicals Agency

EEG

Electroencephalography

EFSA

European Food Safety Authority

EMEA

European Medicines Agency

ETP

European Technology Platform

g

gram

GALT

Gut Associated Lymphoid Tissue

GI

Gastrointestinal

GLP

Good Laboratory Practice

GST

Glutathione-S-transferase

h

hour

HAR

High Aspect Ratio

HARN

High Aspect Ratio Nanoparticles

HPRT

Hypoxanthine-guanine phosphoribosyltransferase

ICRP

International Commission on Radiological Protection

IgG

Immunoglobulin G

IRMM

Institute for Reference Materials and Measurements

ISO

International Organization for Standardisation

JRC

Joint Research Centrekg

kilogram

Kow

Octanol-water partition coefficient

l

litre

LDH

Lactate dehydrogenase2

square metre3mcubic metre

MARCO

Macrophage receptor with collagenous structuremg

milligramm

58

Risk Assessment of Products of Nanotechnologies
minminuteMSDS

Material Safety Data Sheet

MWCNT

Multi-walled Carbon Nanotube

NIOSH

National Institute for Occupational Safety and Health

NIST

National Institute of Standards and Technology

nm

nanometre

NOM

Natural Organic Matter

OECD

Organisation for Economic Cooperation and Development

PAH

Polycyclic Aromatic Hydrocarbons

PBS

Phosphate buffered saline

PEC

Predicted Environmental Concentration

PEG

Polyethylene glycol

PM2.5

Particulate matter below 2.5 µm in diameter (respirable fraction)

QSAR

Quantitative Structure Activity Relationship

REACH

Registration, Evaluation and Authorisation of Chemicals

RM

Reference Material

ROS

Reactive oxygen species

SDS

Sodium dodecyl sulphate

SCCNFP

Scientific Committee on Cosmetic Products and Non-Food Products

SCCP

Scientific Committee on Consumer Products

SCENHIR

Scientific Committee on Emerging and Newly Identified Health Risks

SSA

Specific Surface Area

SWCNT

Single Walled Carbon Nanotube

TG

Test Guideline

TGD

Technical Guidance Document

THF

Tetrahydrofuranµg

microgramµm

micrometre

WWICS

Woodrow Wilson International Centre for Scholars

59

Risk Assessment of Products of Nanotechnologies
7. REFERENCES
Arredouani M, Yang Z, Ning Y, Qin G, Soininen R, Tryggvason K, et al. The scavenger receptor
MARCO is required for lungs defense against Pneumococcal Pneumonia and inhaled particles. J Exp
Med 2004; 200:267-72.
Baalousha M, Manciulea A, Cumberland S, Kendall K, Lead JR. Aggregation and surface propertiesof iron nanoparticles: influence of pH and natural organic matter. Environ Toxicol Chem 2008;
27:1875-82.
Barnes CA, Elsaesser A, Arkusz J, Smok A, Palus J, Leśniak A, et al. Reproducible comet assay of
amorphous silica nanoparticles detects no genotoxicity. Nano Lett 2008; 8:3069-74.
Baun A, Sörensen SN, Rasmussen RF, Hartmann NB, Koch CB. Toxicity and bioaccumulation ofxenobiotic organic compounds in the presence of aqueous suspensions of aggregates of nano-C60.
Aquatic Toxicol 2008; 86:379-87.
Bihari P, Vippola M, Schultes S, Praetner M, Khandoga AG, Reichel CA, et al. Optimized dispersionof nanoparticles for biological in vitro and in vivo studies. Part Fibre Toxicol 2008; 5:14.
Blunk T, Hochstrasser DF, Sanchez JF, Müller BW, Müller RH. Colloidal carriers for intravenous drug
targeting: plasma protein adsorption patterns on surface-modified latex particles evaluated by twodimensional polyacrylamide gel electrophoresis. Electrophoresis 1993; 14:1382-7.
Bopp SK, Lettieri T. Comparison of four different colorimetric and fluorometric cytotoxicity assays ina zebrafish liver cell line. BMC Pharmacol 2008; 8:8.
Borm PJ, Robbins D, Haubold S, Kuhlbusch T, Fissan H, Donaldson K, et al. The potential risk ofnanomaterials: A review carried out for ECETOC. Part Fibre Toxicol 2006; 3:11.
Bouldin JL, Ingle TM, Sengupta A, Alexander R, Hanningan RE, Buchanan RA. Aqueous toxicity andfood chain transfer of quantum dots in freshwater algae and Ceriodaphnia dubia. Environ Toxicol
Chem 2008; 27:1958-63.
Boxall AB, Chaudhry Q, Sinclair C, Jones A, Aitken R, Jefferson B, et al. Current and future
predicted environmental exposure to engineered nanoparticles. DEFRA Report; 2007.
Brayner R. The toxicological impact of nanoparticles. Nano Today 2008; 3:48-55.
Brouwer DH, Gijsbers JH, Lurvink MW. Personal exposure to ultrafine particles in the workplace:
exploring sampling techniques and strategies. Ann Occup Hyg 2004; 48:439-53.
Brown JS, Zeman KL, Bennet WD. Ultrafine particle deposition and clearance in the healthy andobstructed lung. Am J Respir Critic Care Med 2002; 166:1240-7.
Brunauer S, Emmett PH, Teller E. Adsorption of gases in multimolecular layers. J Amer Chem Soc
1938; 60:309-19.
Buford MC, Hamilton RF Jr, Holian A. A comparison of dispersing media for various engineeredcarbon nanoparticles. Part Fibre Toxicol 2007; 4:6.
Cabaleiro-Lago C, Quinlan-Pluck F, Lynch I, Lindman S, Minogue AM, Thulin E, et al. Inhibition ofamyloid protein fibrillation by polymeric nanoparticles. J Am Chem Soc 2008; 130:15437-43.
Cañas JE, Long M, Nations S, Vadan R, Dai L, Luo M, et al. Effects of functionalized andnonfunctionalized single walled carbon nanotubes on root elongation of selected crop species.
Environ Toxicol Chem 2008; 27:1922-31.
Carlson C, Hussain SM, Schrand AM, Braydich-Stolle LK, Hess KL, Jones RL, et al. Inuque cellularinteractions of silver nanoparticles: size dependent generation of reactive oxygen species. J Phys
Chem B 2008; 112,13608-19.
Castro M, Santos MM, Monteiro NM, Vieira N. Measuring lysosomal stability as an effective tool formarine coastal environmental monitoring. Mar Environ Res 2004; 58:741-5.
Cedervall T, Lynch I, Lindman S, Berggård T, Thulin E, Nilson H, et al. Understanding thenanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins fornanoparticles. Proc Natl Acad Sci USA 2007; 104:2050-5.
Chen M, von Mikecz A. Formation of nucleoplasmic protein aggregates impairs nuclear function in
response to SiO2 nanoparticles. Exp Cell Res 2005; 305:51-62.
Chen KL, Elimelech M. Interaction of fullerene (C60) nanoparticles with humic acid and alginate
coated silica surfaces: measurements, mechanisms, and environmental implications. Environ Sci
Technol 2008; 42:7607-14.

60

Risk Assessment of Products of Nanotechnologies
Chen Z, Westerhoff P, Herckes P. Quanitification of C60 fullerene concentrations in water. Environ
Toxicol Chem 2008; 27:1852-9.
Cheng J, Flahaut E, Cheng SH. Effects of carbon nanotubes on developing zebrafish (Danio rerio)
embryos. Environ Toxicol Chem 2007; 26:708-16.
Chien P, Weissman JS, DePace AH. Emerging principles of conformation based prion inheritance.
Ann Rev Biochem 2004; 73:617-56.
Chiti F, Dobson CM. Protein misfolding, functional amyloid, and human disease. Ann Rev Biochem
2006; 75:333-66.
Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, et al. Renal clearance of quantum dots. Nat
Biotechnol 2007; 25:1165-70.
Christian P, Von der Krammer F, Baalousha M, Hofmann T. Nanoparticles: structure, properties,
preparation and behaviour in environmental media. Ecotoxicology 2008; 17:326-43.
Colvin VL. The potential environmental impact of engineered nanomaterials. Nat Biotechnol 2003;
21:1166-70.
Colognato R, Bonelli A, Ponti J, Farina M, Bergamaschi E, Sabbioni E, et al. Comparative
genotoxicity of cobalt nanoparticles and ions on human leukocytes in vitro. Mutagenesis 2008;
23:377-82.
Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD. Gold nanoparticles are taken up by humancells but do not cause acute cytotoxicity. Small 2005; 1:325-7.
Crane M, Handy DR, Garrod J, Owen R. Ecotoxicity test methods and environmental hazard
assessment for engineered nanoparticles. Ecotoxicology 2008; 17:421-37.
Crüts B, Van Etten L, Törnqvist H, Blomberg A, Sandström T, Mills NL, et al. Exposure to diesel
exhaust induces changes in EEG in human volunteers. Part Fibre Toxicol 2008; 5:4.
Davis JM, Addison J, Bolton RE, Donaldson K, Jones AD, Smith T. The pathogenicity of long versus
short fibre samples of amosite asbestos administered to rats by inhalation and intraperitponeal
injection. Br J Exp Pathol 1986; 67:415-30.
De Jong WH, Hagens W, Krystek P, Burger M, Sips A, Geertsma R. Particle size-dependent organ
distribution of gold nanoparticles after intravenous administration. Biomaterials 2008; 29:1912-9.
Des Rieux A, Fievez V, Garinot M, Schneider YJ, Préat V. Nanoparticles as potential oral delivery
systems of proteins and vaccines: a mechanistic approach. J Control Release 2006; 116:1-27.
Dhawan A, Taurozzi JS, Pandey AK, Shan W, Miller SM, Hashsham SA, et al. Stable colloidal
dispersions of C60 fullerenes in water: evidence for genotoxicity. Environ Sci Technol 2006;
40:7394-401.
Donaldson K, Hill IM, Beswick PH. Superoxide anion release by alveolar macrophages exposed torespirable industrial fibres: modifying effect of fibre opsonisation. Exp Toxicol Pathol 1995; 47:22931.
Donaldson K, Tran L, Jimenez LA, Duffin R, Newby DE, Mills N, et al. Combustion-derived
nanoparticles: A review of their toxicology following inhalation exposure. Part Fibre Toxicol 2005;
2:10.
Donaldson K, Aitken R, Tran L, Stone V, Duffin R, Forrest G, et al. Carbon nanotubes: a review of
their properties in relation to pulmonary toxicology and workplace safety. Toxicol Sci 2006; 92:522.
Donaldson K, Stone V, Seaton A, Tran L, Aitken R, Poland C. Re: Induction of mesothelioma in
p53+/- mouse by intraperitoneal application of multi-wall carbon nanotube (Comment on: J Toxicol
Sci 2008; 33:105-16). J Toxicol Sci 2008; 33:385.
Dufour EK, Kumaravel T, Nohynek GJ, Kirkland D, Toutain H. Clastogenicity, photo-clastogenicity or
pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in pre-irradiated or
simultaneously irradiated Chinese hamster ovary cells. Mutat Res 2006; 607:215-24.
Dunford R, Salinaro A, Cai L, Serpone N, Horikoshi S, Hidaka H, et al. Chemical oxidation and DNA
damage catalysed by inorganic sunscreen ingredients. FEBS Lett 1997; 418:87-90.
Dutta D, Sundaram SK, Teeguarden JG, Riley BJ, Fifield LS, Jacobs JM. Adsorbed proteins influenceon biological activity and molecular targeting of nanomaterials. Toxicol Sci 2007; 100:303-15.

61

Risk Assessment of Products of Nanotechnologies
ECETOC. Testing Strategies to Establish the Safety of Nanomaterials—7–8 November 2005,
Barcelona. Workshop Report No. 7. European Centre for Ecotoxicology and Toxicology ofChemicals, Brussels, Belgium; 2006.
EFSA (European Food Safety Authority). Draft Opinion of the Scientific Committee on the risks
arising from nanoscience and nanotechnologies on food and feed safety. Endorsed for public
consultation on 14 October 2008. EFSA, Parma, Italy; 2008.
European Commission. Regulation (EC) No 1907/2006 of the European Parliament, and of theCouncil of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restrictionof Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC
and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as
well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC,
93/105/EC and 2000/21/EC. Official Journal of the European Union L396/1 dd 30.12.2006,
European Commission, Brussels, Belgium; 2006.
European Commission. Communication from the Commission to the European Parliament, theCouncil and the European Economic and Social Committee - Regulatory aspects of nanomaterials,
2008, [SEC(2008) 2036], COM/2008/0366 final. European Commission, Brussels, Belgium; 2008.
Fabian E, Landsiedel R, Ma-Hock L, Wiench K, Wohlleben W, van Ravenzwaay B. Tissue distribution
and toxicity of intravenously administered titanium dioxide nanoparticles in rats. Arch Toxicol
2008; 82:151-7.
Federici G, Shaw BJ, Handy RD. Toxicity of titanium dioxide to rainbow trout (Oncorhynchus
mykiss): gill injury, oxidative stress, and other physiological effects. Aquat Toxicol 2007; 84:41530.
Fernandes TF, Christofi N, Stone V. The environmental implications of nanomaterials. In: MonteiroRiviere NA, Tran CL, editors. Nanotoxicology: characterization, dosing and health effects. Taylor
and Francis, CRC Press, USA; 2007.
Filley TR, Ahn MM, Held BW, Blanchette RA. Investigations of fungal mediated (C60-C70) fullerene
decomposition. Preprints of extended abstracts presented at the 229th ACS National Meeting,
American Chemical Society; 2005 Mar 13-17; San Diego, CA, USA; 45:446-450.
Fisk AT, Norstrom RJ, Cymbalisty CD, Muir DC. Dietary accumulation and depuration ofhydrophobic organochlorines: Bioaccumulation parameters and their relationship with theoctanol/water partition coefficient. Environ Toxicol Chem 1998; 17:951-61.
Floege J, Ehlerding G. Beta-2-microglobulin associated amyloidosis. Nephron 1996; 72: 9-26.
Florence AT, Hillery AM, Hussain N, Jani PU. Factors affecting the oral uptake and translocation ofpolystyrene nanoparticles: histological and analytical evidence. J Drug Target 1995; 3:65-70.
Fortner JD, Lyon DY, Sayes CM, Boyd AM, Falkner JC, Hotze EM, et al. C60 in water: nanocrystal
formation and microbial response. Environ Sci Technol 2005; 39:4307-16.
Franklin N, Rogers N, Apte S, Batley G, Gadd G, Casey P. Comparative toxicity of nanoparticulate
ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): theimportance of particle solubility. Environ Sci Technol 2008; 41:8484-90.
Fujitani Y, Kobayashi T, Arashidani K, Kunugita N, Suemura K. Measurement of the physicalproperties of aerosols in a fullerene factory for inhalation exposure assessment. J Occup Environ
Hyg 2008; 5:380-9.
Geiser M, Rothen-Rutishauser B, Kapp N, Schurch S, Kreyling W, Schulz H, et al. Ultrafine particles
cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells. Environ
Health Perspect 2005; 113:1555-60.
Gonzalez L, Lison D, Kirsch-Volders M. Genotoxicity of engineered nanomaterials: a critical review.
Nanotoxicology 2008; 2:252-73.
Griffitt RJ, Weil R, Hyndman KA, Denslow ND, Powers K, Taylor D, et al. Exposure to copper
nanoparticles causes gill injury and acute lethality in Zebrafish (Danio rerio). Environ Sci Technol
2007; 41:8178-86.
Griffitt RJ, Luo J, Gao J, Bonzongo JC, Barber DS. Effects of particle composition and species on
toxicity of metallic nanomaterials in aquatic organisms. Environ Toxicol Chem 2008; 27:1972-8.
Gurr JR, Wang AS, Chen CH, Jan KY. Ultrafine titanium dioxide particles in the absence ofphotoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology 2005;
213:66-73.

62

Risk Assessment of Products of Nanotechnologies
Hainfeld JF, Slatkin DN, Smilowitz HM. The use of gold nanoparticles to enhance radiotherapy inmice. Phys Med Biol 2004; 49:N309-15.
Handy RD, Eddy FB. Transport of solutes across biological membranes in eukaryotes: anenvironmental perspective. In: van Leeuwen HP, Köster W, editors. Physiological kinetics andtransport at chemical-biological interphases. IUPAC series; John Wiley, Chichester; 2004. p.33756.
Handy RD, Henry TB, Scown TM, Johnston BD, Tyler CR. Manufactured nanoparticles: their uptake
and effects on fish – a mechanistic analysis. Ecotoxicology 2008a; 17:396-409.
Handy RD, von der Kammer F, Lead JR, Hassellöv M, Owen R, Crane M. The ecotoxicology andchemistry of manufactured nanoparticles. Ecotoxicology 2008b; 17:287-314.
Hannah W, Thompson PB. Nanotechnology, risk and the environment: a review. J Environ Monit
2008; 10:291-300.
Hassellöv M, Readman JW, Ranville JF, Tiedje K. Nanoparticle analysis and characterization
methologies in environmental risk assessment of engineered nanoparticles. Ecotoxicology 2008;
17:344-61.
Heckel K, Kiefmann R, Dörger M, Stoeckelhuber M, Goetz AE. Colloidal gold particles as a newmarker of early acute lung injury. Am J Physiol Lung Cell Mol Physiol 2004; 287:L867-78.
Henry TB, Menn FM, Fleming JT, Wilgus J, Compton RN, Sayler GS. Attributing effects of aqueous
C60 nano-aggregates to tetrahydrofuran decomposition products in larval zebrafish by assessmentof gene expression. Environ Health Perspect 2007; 115:1059-65.
Hillyer JF, Albrecht RM. Correlative instrumental neutron activation analysis, light microscopy,transmission electron microscopy, and X-ray microanalysis for qualitative and quantitative
detection of colloidal gold spheres in biological specimens. Microsc Microanal 1998; 4:481-90.
Hillyer JF, Albrecht RM. Gastrointestinal persorption and tissue distribution of differently sized
colloidal gold nanoparticles. J Pharm Sci 2001; 90:1927-36.
Hirsch LR, Stafford RJ, Bankson JA, Sershen SR, Rivera B, Price RE, et al. Nanoshell-mediatednear-infrared thermal therapy of tumors under magnetic resonance guidance. Proc Natl Acad Sci
USA 2003; 100:13549-54.
Holbrook RD, Murphy KE, Morrow JB, Cole KD. Trophic transfer of nanoparticles in a simplified
invertebrate food web. Nat Nanotechnol 2008; 3:352-5.
Hoyt VW, Mason E. Nanotechnology emerging health issues. J Chem Health Safety 2008; 15:10-5.
Hund-Rinke K, Simon M. Ecotoxic effects of photocatalytic active nanoparticles (TiO2) on algae anddaphnids. Environ Sci Pollut Res Int 2006; 13:225-32.
Hussain N, Jani PU, Florence AT. Enhanced oral uptake of tomato lectin-conjugated nanoparticles in
the rat. Pharm Res 1997; 14:613-8.
Hyung H, Fortner JD, Hughes JB, Kim JH. Natural organic matter stabilizes carbon nanotubes in the
aqueaous phase. Environ Sci Technol 2007; 41:179-84.
Ichihara I, Castranova V, Tanioka A, Miyazawa K. Re: Induction of mesothelioma in p53+/- mouseby intraperitoneal application of multi-wall carbon nanotube (Comment on: J Toxicol Sci 2008;
33:105-16). J Toxicol Sci 2008; 33:381-2.
ICRP. Human respiratory tract model for radiological protection. Publication 66. Ann. ICRP 24 (13), Pergamon Press, Oxford, 1994.
Jacobsen NR, Pojana G, White P, Møller P, Cohn CA, Korsholm KS, et al. Genotxicity, cytotoxicity,and reactive oxygen species induced by single-walled carbon nanotubes and C60 fullerenes in the
FE1-MutaTM Mouse lung epithelial cells. Environ Mol Mutagen 2008; 49:476-87.
Jafvert C, Kulkarni P. Buckminsterfullerene’s (C60) octanol-water partition coefficient (Kow) andaqueous solubility. Environ Sci Technol 2008; 42:5945-50.
Jani PU, MacCarthy DE, Florence AT. Titanium dioxide (rutile) particle uptake from the rat GI tract
and translocation to systemic organs after oral administration. Int J Pharm 1994; 105:157-68.
Jemec A, Drobne D, Remskar M, Sepcic K, Tisler T. Effects of ingested nano-sized titanium dioxideon terrestrial Isopods (Porcellio scaber). Environ Toxicol Chem 2008; 27:1904-14.
Ji ZQ, Sun H, Wang H, Xie Q, Liu Y, Wang Z. Biodistribution and tumor uptake of C60(OH)x in mice.
J Nanopart Res 2006; 8:53-63.

63

Risk Assessment of Products of Nanotechnologies
John TA, Vogel SM, Minshall RD, Ridge K, Tiruppathi C, Malik AB. Evidence for the role of alveolar
epithelial gp60 in active transalveolar albumin transport in the rat lung. J Physiol 2001; 533:54759.
John TA, Vogel SM, Tiruppathi C, Malik AB, Minshall RD. Quantitative analysis of albumin uptake
and transport in the rat microvessel endothelial monolayer. Am J Physiol Lung Cell Mol Physiol
2003; 284:L187-96.
Jones RA. Soft condensed matter. Oxford University Press: New York; 2002.
Ju-Nam Y, Lead JR. Manufactured nanoparticles: An overview of their chemistry, interactions andpotential environmental implications. Sci Total Environ 2008; 400:396-414.
Kagan VE, Tyurina YY, Tyurin VA, Konduru NV, Potapovich AI, Osipov AN, et al. Direct and indirect
effects of single walled carbon nanotubes on RAW 264.7 macrophages: role of iron. Toxicol Lett
2006; 165:88-100.
Kane AB. Animal models for malignant mesothelioma. Inhal Toxicol 2006; 18:1001-4.
Kane DB, Oktem B, Johnston MV. Nanoparticle detection by aerosol mass spectrometry. Aerosol Sci
Technol 2001; 34:520-7.
Kang S, Mauter MS, Elimelech M. Physicochemical determinants of multiwalled carbon nanotube
bacterial cytotoxicity. Environ Sci Technol 2008; 42:7528-34.
Kawano T, Yamagata M, Takahishi H, Niidome Y, Yamada S, Katayama Y, et al. Stabilizing ofplasmid DNA in vivo by PEG-modified cationic gold nanoparticles and the gene expression assistedwith electrical pulses. J Control Release 2006; 111:382-9.
Kendall M. Fine airborne urban particles (PM 2.5) sequester lung surfactant and amino acids fromhuman lung lavage. Am J Physiol Lung Cell Mol Physiol 2007; 293:L1053-8.
Kennedy AJ, Hull MS, Steevens JA, Dontsova KM, Chappell MA, Gunter JC, et al. Factors influencing
the partioning and toxicity of nanotubes in the aquatic environment. Environ Toxicol Chem 2008;
27:1932-41.
Kim H, Liu X, Kobayashi T, Kohyama T, Wen FQ, Romberger DJ, et al. Ultrafine carbon black
particles inhibit human lung fibroblast-mediated collagen gel contraction. Am J Resp Cell Mol Biol
2003; 28:111-21.
Kim YS, Kim JS, Cho HS, Rha DS, Kim JM, Park JD, et al. Twenty-eight-day oral toxicity,
genotoxicity, and gender-related tissue distribution of silver nanoparticles in Sprague-Dawley rats.
Inhal Toxicol 2008; 20:575-83.
Klaine SJ, Avarez PJ, Batley GE, Fernandes TF, Handy RD, Lyon DY, et al. Nanomaterials in theenvironment: behavior, fate, bioavailability, and effects. Environ Toxicol Chem 2008; 27:1825-51.
Kocbach A, Herseth JI, Låg M, Refsnes M, Schwarze PE. Particles from wood smoke and traffic
induce differential pro-inflammatory response patterns in co-cultures. Toxicol Appl Pharmacol
2008; 232:317-26.
Koo EH, Lansbury PT, Kelly JW. Amyloid diseases: abnormal
neurodegeneration. Proc Natl Acad Sci USA 1999; 96:9989-90.
Koziara JM, Oh JJ, Akers WS, Ferraris SP, Mumper RJ. Blood
alcohol/polysorbate-based nanoparticles. Pharm Res 2005; 22:1821-8.

proteinaggregationcompatibility

of

in

cetyl

Kreuter J, Shamenkov D, Petrov V, Ramge P, Cychutek K, Koch-Brandt C, et al. Apolipoproteinmediated transport of nanoparticle-bound drugs across the blood-brain barrier. J Drug Target
2002; 10:317-25.
Kreyling W, Scheuch G. Clearance of particles deposited in the lungs. In: Heyder J, Gehr P, editors.
Particle lung interactions. New York, USA: Marcel Dekker; 2000. p.323-76.
Kreyling WG, Semmler M, Erbe F, Mayer P, Takenaka S, Schulz H. Translocation of ultrafine
insoluble particles from lung epithelium to extrapulmonary organs in size dependent but very low. J
Toxicol Environ Health A 2002; 65:1513-30.
Kuhlbusch TA, Neumann S, Fissan H. Number size distribution, mass concentration, and particle
composition of PM1, PM2.5, and PM10 in bag filling areas of carbon black production. J Occup
Environ Hyg 2004; 10:660-71.
Kuhlbusch TA, Fissan H. Particle characteristics in the reactor and pelletizing areas of carbon black
production. J Occup Environ Hyg 2006; 3:558-67.

64

Risk Assessment of Products of Nanotechnologies
Kuhlbusch TA, Fissan H, Asbach C. Measurement and detection of nanoparticles in theenvironment. In: Krug H, editor. Nanotechnology, Volume 2: Environmental Aspects. Wiley-VCH;
2008a. p.229-265.
Kuhlbusch TA, Fissan H, Asbach C. Nanotechnologies and Environmental Risks: Measurement
Technologies and Strategies. In: Linkov I, Steevens J, editors. Nanotechnology: Risk and Benefit.
Springer, Dordrecht, The Netherlands; 2008b. p.235-47, in press.
Kwon JT, Hwang SK, Jin H, Kim DS, Minai-Tehrani A, Yoon HJ, et al. Body distribution of inhaled
fluorescent magnetic nanoparticles in the mice. J Occup Health 2008; 50:1-6.
Labarre D, Vauthier C, Chauvierre C, Petri B, Muller RH. Interactions of blood proteins withpoly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush. Biomaterials
2005; 26:5075-84.
Landsiedel R, Kapp MD, Schulz M, Wiench K, Oesch F. Genotoxicity investigations on
nanomaterials: Methods, preparation and characterization of test material, potential artifacts andlimitations – many questions, some answers. Mutat Res 2008; Nov 11. [Epub ahead of print].
Lead JR, Wilkinson KJ. Natural aquatic colloids: current knowledge and future trends. Environ Chem
2006; 3:159-71.
Lee KL, Nallathamby PD, Browning LM, Osgood CJ, Xu XH. In vivo imaging of transport andbiocompatibility of single silver nanoparticles in early development of zebrafish embryos. ACS Nano
2007; 1:133-43.
Lee WM, An YJ, Yoon H, Kweon HS. Toxicity and bioavailability of copper nanoparticles to theterrestrial plants mung bean (Phaseolus radiatus) and wheat (Triticum aestivum): plant agar test
for water-insoluble nanoparticles. Environ Toxicol Chem 2008; 27:1915-21.
Li N, Sioutas C, Cho A, Schmitz D, Misra C, Sempf J, et al. Ultrafine particulate pollutants induce
oxidatiove stress and mitochondrial damage. Environ Health Perspect 2003; 111:455-60.
Li D, Lyon DY, Li Q, Alvarez P.J. Effect of soil sorption and aquatic natural organic matter on the
antibacterial activity of a fullerene water suspension. Environ Toxicol Chem 2008; 27:1888-94.
Limbach LK, Wick P, Manser P, Grass RN, Bruinink A, Stark WJ. Exposure of engineered
nanoparticles to human lung epithelial cells: influence of chemical composition and catalytic activity
on oxidative stress. Environ Sci Technol 2007; 41:4158-63.
Lin D, Xing B. Root uptake and phytotoxicity of ZnO nanoparticles. Environ Sci Technol 2008;
42:5580-5.
Lindfors K, Kalkbrenner T, Stoller P, Sandoghdar V. Detection and spectroscopy of gold
nanoparticles using supercontinuum white light confocal microscopy. Phys Rev Lett 2004;
93:037401.
Linkov I, Satterstrom FK, Steevens J, Ferguson E, Pleus RC. Multi-criteria decision analysis andenvironmental risk assessment for nanomaterials. J Nanoparticle Res 2007; 9:543-54.
Linse S, Cabaleiro-Lago C, Xue WF, Lynch I, Lindman S, Thulin E, et al. Nucleation of protein
fibrillation by nanoparticles. Proc Natl Acad Sci USA 2007; 104:8691-6.
Liu YY, Liu TY, Chen SY, Liu DM. Synthesis and characterization of nanoporous SiO(2)/pHEMA
biocomposites. J Mater Sci Mater Med 2008; 19:2903-11.
Loo C, Lin A, Hirsch L, Lee MH, Barton J, Halas N, et al. Nanoshell-enabled photonics-based
imaging and therapy of cancer. Technol Cancer Res Treat 2004; 3:33-40.
Lovern SB, Klaper R. Daphnia magna mortality when exposed to titanium dioxide and fullerene(C60) nanoparticles. Environ Toxicol Chem 2006; 25:1132-7.
Lundqvist M; Sethson I; Jonsson BH. Protein adsorption onto silica nanoparticles: conformationalchanges depend on particles curvature and protein stability. Langmuir 2004; 20:10639-47.
Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA. Nanoparticle size and surface
properties determine the protein corona with possible implications for biological impacts. Proc Natl
Acad Sci USA 2008; 105:14265-70.
Luoma SN. Silver Nanotechnologies and the environment: old problems or new challenges? Project
on emerging technologies (PEN) 15. Pew Charitable Trusts, Woodrow Wilson International Center
for Scholars; 2008.
Lyklema J. Pair Interactions. In: Lyklema J, editor. Fundamentals of interface and colloid science,
Volume IV, Particulate colloids. Elsevier Academic Press, Amsterdam; 2005.

65

Risk Assessment of Products of Nanotechnologies
Lynch I, Dawson KA. Protein-nanoparticle interactions. Nano Today 2008; 3:40-7.
Lynch I, Dawson KA, Linse S. Detecting cryptic epitopes created by nanoparticles. Sci STKE 2006;
327:pe14.
Lyon DY, Adams LK, Falkner JC, Alvarez PJ. Antibacterial acitivity of fullerene water suspensions:effects of preparation method and particle size. Environ Sci Technol 2006; 40:4360-6.
Lyon DY, Alvarez P.J. Fullerene water suspension (nC60) exerts antibacterial effects via ROSindependent protein oxidation. Environ Sci Technol 2008; 42:8127-32.
Maynard AD, Baron PA, Foley M, Shvedova AA, Kisin ER, Castranova V. Exposure to carbon
nanotube material: aerosol release during the handling of unrefined single-walled carbon nanotube
material. J Toxicol Environ Health A 2004; 67:87-107.
Miller BG, Searl A, Davis JM, Donaldson K, Cullen RT, Bolton RE, et al. Influence of fibre length,
dissolution and biopersistence on the production of mesothelioma in the rat peritoneal cavity. Ann
Occup Hyg 1999; 43:155-66.
Mills NL, Amin N, Robinson SD, Anand A, Davies J, Patel P, et al. Do inhaled carbon nanoparticles
translocate directly into the circulation in humans? Am J Resp Crit Care Med 2006; 173:426-31.
Mills NL, Donaldson K, Hadoke PW, Boon NA, MacNee W, Cassee FR, et al. Adverse cardiovasculareffects of air pollution. Nat Clin Pract Cardiovasc Med 2009; 6:36-44.
Möller W, Felten K, Sommerer K, Scheuch G, Meyer G, Meyer P, et al. Deposition, retention, andtranslocation of ultrafine particles from the central airways and lung periphery. Am J Respir Crit
Care Med 2008; 177:366-7.
Mordas G, Sipilä M, Kulmala M. Nanoparticle Detection Using Nucleation Regime of the CPC. Book
Nucleation and Atmospheric Aerosols: Springer; Netherlands, 2008. DOI 10.1007/978-1-40206475-3 Copyright 2008 ISBN 978-1-4020-6474-6 (Print) 978-1-4020-6475-3 (Online) Part I DOI
10.1007/978-1-4020-6475-3_43. p.209-13.
Mori T, Takada H, Ito S, Matsubayashi K, Miwa N, Sawaguchi T. Preclinical studies on safety offullerenen upon acute oral administration and evaluation for no mutagenesis. Toxicology 2006;
225:48-54.
Mroz RM, Schins RP, Li H, Jimenez LA, Drost EM, Holownia A, et al. Nanoparticle-driven DNA
damage mimics irradiation-related carcinogenesis pathways. Eur Respir J 2008; 31:241-51.
Mueller N, Nowack B. Exposure modeling of engineered nanoparticles in the environment. Environ
Sci Technol 2008; 42:4447-53.
Murasov V. Comments on "Particle surface characteristics may play important role in phytotoxicityof alumina nanoparticles". Toxicol Lett 2006; 164:185-7.
Murr LE, Bang JJ, Esquivel EV, Guerrero PA, Lopez D.A. Carbon nanotubes, nanocrystal forms, andcomplex nanoparticle aggregates in common fuel-gas combustion sources and the ambient air. J
Nanoparticle Res 2004; 6:241-51.
Murr LE, Guerrero PA. Carbon nanotubes in wood soot. Atmos Sci Lett 2006; 7:93-5.
Nakagawa Y, Wakuri S, Sakamoto K, Tanaka N. The photogenotoxicity of titanium dioxide particles.
Mutat Res 1997; 394:125-32.
Navarro E, Baun A, Behra R, Hartmann NB, Filser J, Miao AJ, et al. Environmental behavior andecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 2008a; 17:37286.
Navarro E, Piccapietra F, Wagner B, Marconi F, Kaegi R, Odzak N, et al. Toxicity of Silver
Nanoparticles to Chlamydomonas reinhardtii. Environ Sci Technol 2008b; 42:8959-64.
Nel A, Xia T, Madler L, Li N. Toxic potential of materials at the nanolevel. Science 2006; 311:622-7.
Nemmar A, Hoet PH, Vanquickenborne B, Dinsdale D, Thomeer M, Hoylaerts MF, et al. Passage of
inhaled particles into the blood circulation in humans. Circulation 2002; 105:411-4.
Nielsen HD, Berry LS, Stone V, Fernandes TF. Interactions between carbon black nanoparticles andthe brown algae Fucus serratus: inhibition of fertilization and zygotic development. Nanotoxicology
2008; 2:88-97.
Niidome T, Yamagata M, Okamoto Y, Akiyama Y, Takahishi H, Kawano T, et al. PEG-modified gold
nanorods with a stealth character for in vivo application. J Control Release 2006; 114:343-7.

66

Risk Assessment of Products of Nanotechnologies
Niwa Y, Iwai N. Nanomaterials induce oxidized low-density lipoprotein cellular uptake inmacrophages and platelet aggregation. Circ J 2007; 71:437-44.
Noble CA, Prather KA. Real-time single particle mass spectrometry: a historical review of a quarter
century of the chemistry of aerosols. Mass Spectrom Rev 2000; 4:248-74
O’Neal DP, Hirsch LR, Halas NJ, Payne JD, West JL. Photo-thermal tumor ablation in mice usingnear infrared-absorbing nanoparticles. Cancer Lett 2004; 209:171-6.
Oberdörster G, Sharp Z, Atudorei V, Elder A, Gelein R, Lunts A, et al. Exrapulmonary translocationof ultrafine carbon particles following whole body inhalation exposure of rats. J Toxicol Environ
Health Part A 2002; 65:1531-43.
Oberdörster E. Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in brain ofjuvenile largemouth bass. Environ Health Perspect 2004; 112:1058-62.
Oberdörster G, Sharp Z, Atudorei V, Elder A, Gelein R, Kreyling W, et al. Translocation of Inhaled
Ultrafine Particles to the Brain. Inhal Toxicol 2004; 16:437-45.
Oberdörster G, Maynard A, Donaldson K, Castranova V, Fitzpatrick J, Ausman K, et al. Principles forcharacterizing the potential human health effects from exposure to nanomaterials: elements of ascreening strategy. Part Fibre Toxicol 2005a; 2:8.
Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: an emerging discipline evolving form
studies of ultrafine particles. Environ Health Perspect 2005b; 113:823-39.
Oberdörster E, Zhu S, Blickley TM, McClellan-Green P, Haasch ML. Ecotoxicology of carbon-basedengineered nanoparticles: effects of fullerene (C60) on aquatic organisms. Carbon 2006; 44:111220.
OECD. Water Solubility: OECD Test Guideline 105. Organization for Economic Coordination andDevelopment. Paris, France; 1995.
OECD. Bioconcentration: Flow-through Fish Test: OECD Test Guideline 305. Organization for
Economic Coordination and Development. Paris, France; 1996.
OECD. Guidance document on the validation and international acceptance of new or updated test
methods for hazard assessment. OECD Environment, Health and Safety Publications Series on
Testing and Assessment No. 34. Organization for Economic Coordination and Development. Paris,
France; 2005
OECD. List of manufactured nanomaterials and list of endpoints for phase one of the OECD testing
programme. OECD Environment, Health and Safety Publications Series on the safety of
manufactured nanomaterials No 6. Document ENV/JM/MONO(2008)13. Organization for Economic
Coordination and Development. Paris, France; 2008aOECD. Sediment-water Lumbriculus toxicity test using speked sediment. OECD Test Guideline 225.
Organization for Economic Coordination and Development. Paris, France; 2008b.
Oughton DH, Hertel-Aas T, Pellicier E, Mendoza E, Joner EJ. Neutron activation of engineered
nanoparticles as a tool for tracing their environmental fate and uptake in organisms. Environ
Toxicol Chem 2008; 27:1883-7.
Oyewumi MO, Yokel RA, Jay M, Coakley T, Mumper RJ. Comparison of cell uptake, biodistribution
and tumor retention of folate-coated and PEG-coated gadolinium nanoparticles in tumor-bearing
mice. J Control Release 2004; 95:613-26.
Pacurari M, Yin XJ, Zhao J, Ding M, Leonard SS, Schwegler-Berry D, et al. Raw single-wall carbon
nanotubes induce oxidative stress and activate MAPKs, AP-1, NF-kappaB, and Akt in normal andmalignant human mesothelial cells. Environ Health Perspect 2008; 116:1211-7.
Panté N, Kann M. Nuclear pore complex is able to transport macromolecules with diameters of
about 39 nm. Mol Biol Cell 2002; 13:425-34.
Papageorgiou I, Brown C, Schins R, Singh S, Newson R, Davis S, et al. The effects of nano- andmicro-sized particles of cobalt-chromium alloy on human fibroblasts in vitro. Biomaterials 2007;
28:2946-58.
Petersen EJ, Huang Q Weber WJ. Ecological uptake and depuration of carbon nanotubes byLumbriculus variegates. Environ Health Perspect 2008a; 116:496-500.
Petersen EJ, Huang Q, Weber WJ Jr. Bioaccumulation of radio-labeled carbon nanotubes by Eisenia
foetida. Environ Sci Technol 2008b; 42:3090-5.

67

Risk Assessment of Products of Nanotechnologies
Poland C, Duffin R, Kinloch I, Maynard A, Wallace W, Seaton A, et al. Carbon nanotubes introduced
into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat
Nanotechnol 2008; 3:423-8.
Pott F. Detection of mineral fibre carcinogenicity with the intraperitoneal test. Recent results andtheir validity. Ann Occup Hyg 1995; 39:771-9.
Quik JT, Lynch I, Salvati A, Miermans K, Van de Meent D. Behavior of ceriumdioxide nanoparticles
under environmentally relevant conditions. Submitted to Environ Sci Technol 2008.
Radomski A, Jurasz P, Alonso-Escolano D, Drews M, Morandi M, Malinski T, et al. Nanoparticleinduced platelet aggregation and vascular thrombosis. Br J Pharmacol 2005; 146:882-93.
Radt B, Smith TA, Caruso F. Optically addressable nanostructured capsules. Adv Mater 2004;
16:2184-9.
Rahman Q, Lohani M, Dopp H, Pemsel L, Jonas D, Weiss G. Evidence that ultrafine titanium dioxide
induces micronuclei and apoptosis in syrian hamster embryo fibroblasts. Environ Health Perspect
2002; 110:797-800.
Reeves JF, Davies SJ, Dodd NJ, Jha AN. Hydroxyl radicals (y OH) are associated with titanium
dioxide (TiO2) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells. Mutat Res
2008; 640:113-22.
Roberts A, Mount AS, Seda B, Souther J, Qiao R, Lin S, et al. In vivo biomodification of lipid-coatedcarbon nanotubes by Daphnia magna. Environ Sci Technol 2007; 41:3025-9.
Rosenkranz P, Stone V, Chaudry Q, Fernandes TF. A comparison of nanoparticle and fine particle
uptake by Daphnia magna. Environ Toxicol Chem 2009. In Press.
Royal Society and Royal Academy of Engineering. Nanoscience and nanotechnologies: opportunities
and uncertainties. UK; July 2004.
Sadauskas E, Wallin H, Stoltenberg M, Vogel U, Doering P, Larsen A, et al. Kupffer cells are central
in the removal of nanoparticles from the organism. Part Fibre Toxicol 2007; 4:10.
Sager TM, Porter DW, Robinson VA, Lindsley WG, Schwegler-Berry DE, Castranova V. Improved
method to disperse nanoparticles for in vitro and in vivo investigation of toxicity. Nanotoxicology
2007; 1:118-29.
Saleh NB, Pfefferle LD, Elimelech M. Aggregation kinetics of multiwalled carbon nanotubes inaquatic systems: measurements and environmental implications. Environ Sci Technol 2008;
42:7963-9.
Sayes CM, Reed KL, Warheit DB. Assessing toxicity of fine and nanoparticles: Comparing in vitro
measurements to in vivo pulmonary toxicity profiles. Toxicol Sci 2007; 97:163-80.
SCCNFP. Opinion of the Scientific Committee on Cosmetic Products and Non-Food Products
Intended for Consumers Concerning Titanium Dioxide. 2000. Available from: URL:
http://ec.europa.eu/health/ph_risk/committees/04_sccp/04_sccp_en.htm
SCCNFP. Opinion of the Scientific Committee on Cosmetic Products and Non-Food Products
IntendedforConsumers
Concerning
Zinc
Oxide.
2003.
Available
from:
URL:
http://ec.europa.eu/health/ph_risk/committees/04_sccp/04_sccp_en.htm
SCENIHR. The appropriateness of existing methodologies to assess the potential risks associatedwith engineered and adventitious products of nanotechnologies. 2006. Available from: URL:
(http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_003b.pdf)
SCENIHR. Opinion on the appropriateness of the risk assessment methodology in accordance withthe technical guidance documents for new and existing substances for assessing the risks ofnanomaterials.
2007a.
Available
from:
URL:
http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_010.pdf
SCENIHR (2007b), Opinion on the scientific aspects of the existing and proposed definitions
relating
toproducts
ofnanoscienceandnanotechnologies,at
http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_012.pdf
Schins RP, Knaapen AM. Genotoxicity of poorly soluble particles. Inhal Toxicol 2007; 19 (Suppl
1):189-98.
Scott-Fordsmand JJ, Krogh PH, Scaefer M, Johansen A. The toxicity testing of double-walled
nanotubes-contaminated food to Eisenia veneta earthworms. Ecotox Environ Safety 2008; 71:6169.

68

Risk Assessment of Products of Nanotechnologies
Semmler M, Seitz J, Erbe F, Mayer P, Heyder J, Oberörster G, et al. Long term clearance kinetics ofinhald ultrafine insoluble iridium particles from the rat lung, including transient translocation into
secondary organs. Inhal Toxicol 2004; 16:453-9.
Semmler-Behnke M, Fertsch S, Schmid O, Wenk A, Kreyling WG. Uptake of 1.4 mm versus 18mm
gld particles by secondary target organs is size dependent in control and pregnants rats after
intratracheal or intravenous application. Proceedings of Euro Nanoforum - Nanotechnology inIndustrial
Applications
2007,
p.102-4.
Available
from:
URL:
http://www.euronanoforum2007.de/download/Proceedings ENF2007.pdf.
Semmler-Behnke M, Kreyling WG, Lipka J, Fertsch S, Wenk A, Takenaka S, et al. Biodistribution of
1.4 nm and 18 nm gold particles in rats. Small 2008; 71:616-9.
Shvedova AA, Kisin ER, Mercer R, Murray AR, Johnson VJ, Potapovich AI, et al. Unusual
inflammatory and fibrogenic pulmonary responses to single walled carbon nanotubes in mice. Am J
Physiol Lung Cell Mol Physiol 2005; 289:L698-708.
Shvedova AA, Fabisiak JP, Kisin ER, Murray AR, Roberts JR, Tyurina YY, et al. Sequential exposure
to carbon nanotubes and bacteria enhances pulmonary inflammation and infectivity. Am J Respir
Cell Mol Biol 2008b; 38:579-90.
Shvedova AA, Kisin ER, Murray AR, Johnson VJ, Gorelik O, Arepalli S, et al. Inhalation versus
aspiration of single walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative
stress and mutagenesis. Am J Physiol Lung Cell Mol Physiol 2008a; 295:L552-65.
Singh R, Pantarotto D, Lacerda L, Pastorin G, Klumpp C, Prato M, et al. Tissue biodistribution andblood clearance rates of intravenously administered carbon nanotube radiotracers. Proc Natl Acad
Sci USA 2006; 103:3357-62.
Smith CJ, Shaw BJ, Handy RD. Toxicity of single walled carbon nanotubes on rainbow trout,(Oncorhynchus mykiss): respiratory toxicity, organ pathologies, and other physiological effects.
Aquat Toxicol 2007; 82:94-109.
Smith JN. Atmospheric nanoparticles: formation and physicochemical properties. In: Schwarz JA,
Contescu CI, Putyera K. Dekker encyclopedia of nanoscience and nanotechnology, 10.1081/E-ENN120023640. 2004.
Sondi I, Salopek-Sondi B. Silver nanoparticles as antimicrobial agent: a case study on E. coli as amodel for Gram-negative bacteria. J Colloid Interface Sci 2004; 275:177-82.
Stapleton HM, Letcher RJ, Li J, Baker JE. Dietary accumulation and metabolism of polybrominateddiphenyl ethers by juvenile carp (Cyprinus carpio). Environ Toxicol Chem 2004; 23:1939-46.
Stolpe B, Hassellöv M. Changes in size distribution of fresh water nanoscale colloidal material andassociated elements on mixing with seawater. Geochim Cosmochim Acta 2007; 71:3292-301.
Stone V, Kinloch I, Clift M, Fernandes TF, Ford A, Christofi N, et al. Nanoparticle toxicology andecotoxicology: the role of oxidative stress. In: Zhao Y, Nalwa HS, editors. Nanotoxicology.
American Scientific Publishers, USA; 2007.
Sweet L, Strohm B. Nanotechnology-life cycle risk management. Human Ecotox Risk Assess 2006;
12:528-51.
Swiss Federal Office of Public Health. Guidelines on the precautionary matrix for synthetic
nanomaterials.
Bern,
Switzerland;
2008.
Available
from:
URL:
http://www.bag.admin.ch/themen/chemikalien/00228/00510/05626/index.html?lang=en
Takagi A, Hirose A, Nishimura T, Fukomori N, Ogata A, Ohashi N, et al. Induction of mesothelioma
in p53+/- mouse by intraperitoneal application of multiwall carbon nanotube. J Toxicol Sci 2008;
33:105-16.
Takahashi S, Matsuoka O. Cross placental transfer of 198Au-colloid in near term rats. J Radiat Res(Tokyo) 1981; 22:242-9.
Takenaka S, Karg E, Kreyling WG, Lentner B, Möller W, Behnke-Semmler M, et al. Distribution
pattern of inhaled ultrafine gold particles in the rat lung. Inhal Toxicol 2006; 18:733-40.
Theogaraj E, Riley S, Hughes L, Maier M, Kirkland D. An investigation of the photo-clastogenic
potential of ultrafine titanium dioxide particles. Mutat Res 2007; 634:205-19.
Tiede K, Boxall A, Tear S, Lewis J, David H, Hassellöv M. Detection and characterization ofengineered nanoparticles in food and the environment. Food Addit Contam Part A Chem Anal
Control Expo Risk Assess 2008; 25:795-821.

69

Risk Assessment of Products of Nanotechnologies
Tsai SJ, Ashter A, Ada E, Mead JL, Barry CF, Ellenbecker MJ. Airborne nanoparticle releaseassociated with the compounding of nanocomposites using nanoalumina as fillers. Aerosol and Air
Quality Res 2008; 8:160-77.
Van Dijk MA, Tchebotareva AL, Orrit M, Lippitz M, Berciaud S, Lasne D, et al. Absorption andscattering microscopy of single metal nanopartcles. Phys Chem Chem Phys 2006; 8:3486-95.
Van Hoecke K, de Schamphelaere KA, van der Meeren P, Lucas SP, Janssen CR. Ecotoxicity of silica
nanoparticles to the green alga Pseudokirchneriella subcapitata: importance of surface area.
Environ Toxicol Chem 2008; 27:1948-57.
Vevers WF, Jha AN. Genotoxic and cytotoxic potential of titanium dioxide (TiO2) nanoparticles onfish cells in vitro. Ecotoxicology 2008; 17:410-20.
Von Gleich A, Steinfeldt M, Petschow U. A suggested three-tiered approach to assessing theimplications of nanotechnology and influencing its development. J Cleaner Prod 2008; 16:899-909.
Wake DD, Mark C. Northage: ultrafine aerosols in the workplace. Ann Occup Hyg 2002; 46 (Suppl
1):235-8.
Wallace WE, Keane MJ, Gautam, Shi XC, Murray D, Ong T. Dispersion of nanoparticles inpulmonary surfactants for in vitro toxicity studies: Lessons from ultrafine diesel exhaust particles
and fine dusts. In: Monteiro-Roviere NA, Tran CL, editors. Nanotoxicology: characterization, dosing
and health effects. 2007. p.153-72.
Wang JJ, Sanderson BJ, Wang H. Cyto- and genotoxicity of ultrafine TiO2 particles in cultured
human lymphoblastoid cells. Mutat Res 2007; 628:99-106.
Wardak A, Gorman ME, Swami N, Deshpande S. Identification of risks in the life cycle ofnanotechnology-based products. J Ind Ecol 2008; 12:435-48.
Warheit DB, Carakostas MC, Bamberger JR, Hartsky MA. Complement facilitates macrophage
phagocytosis of inhaled iron particles but has little effect in mediating dilica induced lunginflammatory and clearance responses. Environ Res 1991; 56:186-203.
Warheit DB, Reed KL, Webb TR. Pulmonary toxicity studies in rats with triethoxyoctylsilane (OTES)coated, pigment grade titanium dioxide particles: bridging studies to predict inhalation hazard. Exp
Lung Res 2003; 29:593-606.
Warheit DB, Hoke RA, Finlay C, Donner EM, Reed KL, Sayes CM. Development of a base set of
toxicity tests using ultrafine TiO2 particles as a component of nanoparticle risk assessment. Toxicol
Lett 2007; 171:99-110.
Wick P, Manser P, Limbach LK, Dettlaff-Weglikowska U, Krumeich F, Roth S, et al. The degree andkind of agglomeration affect carbon nanotube cytotoxicity. Toxicol Lett 2007; 168:121-31.
Wiebert P, Sanchez-Crespo A, Falk R, Philipson K, Lundin A, Larsson S, et al. No significant
translocation of inhaled 35-nm carbon particles to the circulation in humans. Inhalation Toxicol
2006a; 18:741-7.
Wiebert P, Sanchez-Crespo A, Seitz J, Falk R, Philipson K, Kreyling WG, et al. Negligible clearanceof ultrafine particles retained in healthy and affected human lungs. Eur Respir J 2006b; 28:286-90.
Wiesner MR. Responsible development of nanotechnologies for water and wastewater treatment.
Water Sci Technol 2006; 53:45-51.
Wijnhoven S, Herberts C, Hagens W, Oomen A, Heugens E, Roszek B, et al. Nano-silver. A review
of available data and knowledge gaps. Report 360003001/2008 National Institute for Public Healthand the Environment (RIVM), Bilthoven, the Netherlands; 2009.
Wu WH, Sun X, Yu YP, Hu J, Zhao L, Liu Q, et al. TiO2 nanoparticles promote beta-amyloid
fibrillation in vitro. Biochem Biophys Res Comm 2008; 373:315-8.
WWICS. Woodrow Wilson International Centre for Scholars: Project on emerging nanotechnologies,
Consumer product Inventory. Washington, USA; 2008. [Accessed 1st October 2008] Available from:
URL: http://www.nanotechproject.org/inventories/consumer/
Xia T, Korge P, Weiss JN, Li N, Venkatesen MI, Sioutas C, et al. Quinones and aromatic chemicalcompounds in particulate matter induce mitochondrial dysfunction: implications for ultra-fineparticle toxicity, Environ Health Perspect 2004; 112:1347-58.
Yang L, Watts DJ. Particle surface characteristics may play important role in phytotoxicity ofalumina nanoparticles. Toxicol Lett 2005; 158:122-32.

70

Risk Assessment of Products of Nanotechnologies
Yang H, Liu Ch, Yang D, Zhang H, Xi Z. Comparative study of cytotoxicity, oxidative stress andgenotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition.
J Appl Toxicol 2009; 29:69-78.
Yeganeh B, Kull CM, Hull MS, Marr LC. Characterization of airborne particles during production ofcarbonaceous nanomaterials. Environ Sci Technol 2008; 42:4600-6.
Yu LE, Yung LY, Ong CN, Tan YL, Balasubramaniam S, Hartono D, et al. Translocation and effects ofgold nanoparticles after inhalation exposure in rats. Nanotoxicology 2007; 1:235-42.
Zakharenko LP, Zakharov IK, Vasyunina EA, Karamysheva TV, Danilenko AM, Nikiforov AA.
Determination of fullerene C60 and fullerol by the somatic mutation and recombination test in
Drosophila melanogaster and SOS Chromotest. Genetica 1997; 33:405-9.
Zhang X, Sun H, Zhang Z, Niu Q, Chen Y, Crittenden JC. Enhanced bioaccumulation of cadmium incarp in the presence of titanium dioxide nanoparticles. Chemosphere 2007; 67:160-6.
Zhang LW, Yu WW, Colvin VL, Monteiro-Riviere NA. Biological interactions of quantum dot
nanoparticles in skin and in human epidermal keratinocytes. Toxicol Appl Pharmacol 2008;
228:200-11.
Zhu S, Obördorster E, Haasch ML. Toxicity of an engineered nanoparticle (fullerene, C60) in two
aquatic species, Daphnia and fathead minnow. Mar Environ Res 2006; 62 (Suppl):S5-9.
Zhu X, Zhu L, Li Y, Duan Z, Chen W, Alvarez PJ. Developmental in Zebra fish (Danio rerio) embryos
after exposure to manufactured nanomaterials: buckminsterfullerene aggregates (n C60) andfullerol. Environ Toxicol Chem 2007; 26:976-9.

71